HBV in-vitro drug screening model and application thereof

Abstract

The invention belongs to the field of molecular biology, and discloses an HBV (hepatitis B virus) in-vitro drug screening model and application thereof, and the HBV in-vitro drug screening model comprises nucleic acid molecules, plasmids and cells of HBV B2 and C2 reference genomes with Chinese population infection representativeness. The nucleic acid molecule, the plasmid and the cell can represent HBV infected genome sequence B2 type and C2 type of Chinese population, most mutation conditions are effectively covered, and a powerful tool is provided for screening drugs aiming at HBV viruses.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and relates to a screening model for HBV drugs. Background Art

[0002] HBV (Hepatitis B Virus) is a hepatotropic virus belonging to the Hepadnaviridae family. Its genome is composed of double-stranded, circular, and incompletely closed DNA. Due to the spontaneous error rate and lack of proofreading activity of the HBV polymerase, the virus has numerous subtypes, primarily classified into 10 genotypes, A, J, and C. The predominant genotypes prevalent in different regions vary significantly. According to one study, the predominant subtypes in China are genotype B (28.15%) and genotype C (64.35%). Genotypes can also be further divided into subtypes based on genomic differences greater than 4%. Currently, at least 35 subtypes have been identified. As HBV genomic data accumulate, distinct regional preferences have been found among subtypes, and these subtypes may differ in their natural history of infection, clinical manifestations, and response to antiviral drugs. Epidemiological sampling in China indicates that genotypes B2 and C2 together account for >95% of HBV in the Chinese population.

[0003] HepG2 is a commonly used cell type for transfection with plasmids carrying the HBV genome. Compared with models constructed by using HBV to infect PHH, as a cell source of liver cancer, it has the advantages of stable expression, easy cell acquisition, low cost, batch stability, and strong reproducibility. The model constructed with it is more suitable for large-scale in vitro screening of anti-HBV drugs. Some common HBV in vitro screening models are constructed based on HepG2 cells, such as HepG2.2.15, the only widely available commercial HBV cell model on the market. It is composed of HepG2 cells transfected with a recombinant pDoLTHBV-1 plasmid containing two full HBV-DNA genes connected head to tail. The HBV genome is integrated into the HepG2.2.15 genome, which can asexually reproduce in vitro and stably secrete HBsAg, HBeAg and complete Dane particles in the culture medium for a long time. It can produce HBV mRNA with lengths of 3.5kb, 2.5kb, and 2.1kb. It is the most suitable cell for in vitro screening of anti-HBV drugs, HBV replication and hepatocyte response to interferon (IFN). However, the HBV genotype inserted in HepG2.2.15 still belongs to an isolate of type D, and the reference genome genbank number of the strain is U95551. Current in vitro models are constructed from a single HBV genome isolated from a single patient, and therefore cannot simulate the behavior of most strains within a population. For high-throughput screening of small nucleic acid drugs that silence HBV mRNA expression through nucleic acid-specific reverse complementation, results from a single HBV genome cannot adequately characterize the behavior of HBV populations with regional preferences. Summary of the Invention

[0004] The main purpose of the present invention is to assemble and construct HBV B2 and C2 reference genome sequences representative of the infection in the Chinese population through analysis of HBV sequences in a large number of Chinese populations; to enhance the expression of relevant viral components of HBV plasmid vectors in HepG2 cells by optimizing the breakpoints and the positions of repeated fragments, and to establish an in vitro screening model for anti-hepatitis B drugs for the Chinese population, so that the model has important practical value for screening gene therapy drugs with specific targeting sequences.

[0005] In a first aspect, the present invention provides an isolated nucleic acid molecule, the nucleic acid sequence of which is shown in SEQ ID NO: 3 or 4. The nucleic acid molecule represents the B2 or C2 HBV genome of the Chinese population.

[0006] In a second aspect, the present invention provides a plasmid comprising the nucleic acid molecule of the present invention; preferably, the starting plasmid of the plasmid is pcDNA3.1(+).

[0007] Preferably, the plasmid comprises 1.3 times the length of the nucleic acid molecule of the present invention.

[0008] Further preferably, the plasmid is B2-pcDNA3.1 or C2-pcDNA3.1, wherein the nucleic acid sequence of B2-pcDNA3.1 is shown in SEQ ID NO: 5, and the nucleic acid sequence of C2-pcDNA3.1 is shown in SEQ ID NO: 6.

[0009] In a third aspect, a cell is provided, wherein the cell comprises the nucleic acid molecule or plasmid described above in the present invention.

[0010] Preferably, the host cell of the cell is HepG2 cell.

[0011] In a fourth aspect, a kit is provided for screening HBV drugs, which comprises the nucleic acid molecule, plasmid or cell of the present invention.

[0012] In a fifth aspect, a model for screening HBV drugs is provided, comprising the nucleic acid molecule, plasmid, cell or kit of the present invention. Preferably, the model is selected from the nucleic acid molecule, plasmid, cell or kit of the present invention.

[0013] The sixth aspect provides the use of the nucleic acid molecules, plasmids, cells, kits or models of the present invention in screening HBV drugs.

[0014] In a seventh aspect, a method for screening HBV drugs is provided, comprising using the nucleic acid molecules, plasmids, cells, kits or models of the present invention.

[0015] Preferably, the screening method comprises: culturing the cells of the present invention, administering the HBV drug to be screened, and detecting cell expression.

[0016] This invention, through bioinformatics analysis combined with biological experimental validation, has constructed an in vitro drug screening model for HBV infection in the Chinese population. This model represents the genome sequences of HBV-infected genotypes B2 and C2 in the Chinese population, effectively covering the majority of mutations, and provides a powerful tool for drug screening against HBV infection in the Chinese population. It should be noted that this invention is funded by the Sichuan Provincial Science and Technology Plan (Project No. 2022JDRC0050). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : R between two SNPs in the population at the B2 locus 2 Value distribution;

[0018] Figure 2 : R between two SNPs in the C2 locus 2 Value distribution;

[0019] Figure 3 : Sequencing results of B2 reference sequence;

[0020] Figure 4 : Sequencing results of C2 reference sequence;

[0021] Figure 5 : Standard curve of HBeAg protein expression;

[0022] Figure 6 :HBsAg protein expression standard curve;

[0023] Figure 7 : Inhibitory effect of the compound on B2-HepG2 HBV RNA;

[0024] Figure 8 : Inhibitory effect of the compounds on C2-HepG2 HBV RNA;

[0025] Figure 9 : The inhibitory effect of the compound on B2-HepG2 HBV HBsAg protein;

[0026] Figure 10 : The inhibitory effect of the compounds on C2-HepG2 HBV HBsAg protein. DETAILED DESCRIPTION

[0027] Based on the regional distribution of HBV subtypes, the present invention collected, organized, and classified the genome sequences of HBV subtypes in China. Two HBV genome sequences were constructed that characterize infection in the Chinese population. Based on these sequences, 1.3 copies of the HBV genome were integrated into a plasmid vector, which was then transformed into HepG2 cells to construct an in vitro anti-HBV drug screening model that normally transcribes and expresses HBV-related components. This model significantly fills the gap in screening nucleic acid-based therapeutic drugs for HBV virus characteristics in China.

[0028] The experimental methods used in the following examples are routine operations in the field of biology, and all bioinformatics analyses were completed under the Ubuntu 22.04 system.

[0029] Unless otherwise specified, the reagents and instruments used in the following examples are conventional reagents and instruments and can be obtained through commercial channels.

[0030] Example 1: Acquisition of B2 and C2 HBV genomes from the Chinese population

[0031] Genome sequences of genotypes B and C were isolated from the HBVdb database (genbank accession number AB073853 for genotype B and X75665 for genotype C). For ease of writing and coordinate positioning, the starting coordinates of all circular HBV DNA linear reference genomes were set to the commonly used EcoR1 restriction site, with the coordinate of this site defined as 1. Based on this, genotype B and C sequences were further subtyped, with subtypes classified according to subtype differentiation (i.e., inter-genotype differences >4%). Clustering at 96% similarity was performed using CD-HIT. Based on the subtype classification results, 1636 genotype C2 sequences were identified from 3070 genotype C sequences, with C2 accounting for approximately 53% of genotype C sequences. Furthermore, 1416 genotype B2 sequences were identified from 2069 genotype B sequences, with B2 accounting for approximately 68% of genotype B sequences.

[0032] Among the clustering results of C2 type, its reference sequence is isolated from Taiwanese people with GenBank number AY167091, and other known C2 types such as GenBank number GQ377617 (isolated from Chinese people) are also clustered in this category.

[0033] In the clustering results of type B2, the reference sequence of type B2 is the genbank number KU964282 isolated from Shanghai, China. Other known B2 types such as the genbank number GU815637 (isolated from Chinese) are also clustered in this category.

[0034] The present invention uses a custom Python script program to pull the annotation information of all B2 type sequences in NCBI nucleotide and performs statistical analysis on their sequence sources. A total of 1369 sequences with regional annotation information are captured, of which 1227 (90%) sequences are from China, and most of the other sequences are from other regions in East Asia.

[0035] At the same time, a script program was used to pull the annotation information of all C2-type sequences in NCBI nucleotide, and a statistical analysis of their sequence sources was performed. A total of 1,388 sequences were annotated, of which 1,271 (92%) sequences were from China, and most of the other sequences were from other regions in East Asia.

[0036] A collection of B2 and C2 genome sequences with regional information was obtained, of which >90% were from the Chinese population (at least 90% for the B2 type collection and at least 92% for the C2 type collection). In addition, due to the lack of annotation of some sequences, in order to maximize the characterization of HBV sequences in the Chinese population, the present invention included all B2 and C2 sequences obtained by typing in the analysis. From a sampling perspective, it can ensure that >90% of the HBV genome sequences are from the Chinese population, and the remaining ≤10% of the sequences are basically from East Asians within the B2 and C2 types. Therefore, the data set of B2 and C2 sequences isolated and obtained by the present invention can characterize the situation of HBV in the Chinese population.

[0037] Example 2: Locus base distribution statistics of the B2 and C2 HBV genome sequence sets of the Chinese population and their maximum probability genome sequence construction

[0038] The present invention used the MAFFT software to perform multiple alignment analysis on a collection of 1,416 B2 HBV genome sequences and a collection of 1,636 C2 HBV genome sequences. Based on the results of the multiple alignment analysis, the base distribution at each locus in the B2 and C2 sequence collections was statistically analyzed. The distribution of bases at each locus was statistically analyzed to determine the maximum allele. 97.16% of the major allele sites at the corresponding loci in the C2 sequence collection had a conservation rate greater than 90% within the population, with only 0.52% (17) of the gene coordinate sites having a conservation rate less than 50%. 98.43% of the major allele sites at the corresponding loci in the B2 sequence collection had a conservation rate greater than 90% within the population, with only 0.15% (5) of the gene coordinate sites having a conservation rate less than 50%. Based on the statistical analysis results of this multiple alignment, the present invention constructed the maximum probability assembled genotypes of the Chinese population B2 and C2 based on the largest allele, SEQ ID NO: 1 and SEQ ID NO: 2, respectively, to characterize the high-probability genotypes of the HBV population in China. In the two sequences, due to the lack of a portion of the HBV genome at some gene coordinate sites, there is a sequence insertion in the middle, but there is a high probability that HBV at this site does not have a base insertion. In order to align all sequence coordinates for convenient statistical analysis, the sequence is represented by N, representing the gap region.

[0039] Example 3: Identification of linked inheritance in the B2 and C2 HBV genome sequence collections of the Chinese population and correction of the most likely genome sequence

[0040] In order to prevent the simultaneous linkage inheritance of two common mutation points in the population, an R 2 The value is used to represent the probability of two mutation points occurring simultaneously in the population being greater than a random combination. Points with a greater probability of two mutation points being inherited simultaneously than a random combination are combined together to modify the maximum probability genomic sequence. The maximum allele in other genomic coordinates remains unchanged, and the gap region in the genomic sequence is removed.

[0041] In the formula, P(A) and P(B) are the sites on two gene coordinates, respectively, indicating the frequency of the base at a certain coordinate appearing in the population, which is achieved by statistically analyzing the distribution of bases at the corresponding gene coordinates in the sequence set; P(AB) represents the frequency of two bases AB at the gene coordinates being linked and co-inherited on the same genome, which is achieved by statistically analyzing the genetic patterns of two points on each sequence in the genome set. 2 The value formula is as follows:

[0042]

[0043] When the denominator is 0, it does not participate in the discussion, which means that the probability of P(A) or P(B) appearing in the group is 100%, and there is no need to study it. 2 When R is equal to 0, the SNPs at the two base sites completely conform to the random combination pattern; when R 2 When it is equal to 1, it means that the SNPs of the two gene loci are linked. In view of this idea, the present invention customizes a Python script program to calculate the R between two points at the locus where the major allele population frequency is ≤80%. 2 value.

[0044] The results of statistical analysis of linkage inheritance in the B2 and C2 sequence sets showed that most of the sites were in line with the random inheritance model. Figure 1 、 Figure 2 (The horizontal axis is R 2 Value, the vertical axis represents R 2 However, there are cases of linked inheritance between individual sites (see Tables 1 and 2), where the coordinates of the associated site start with 0.

[0045] The present invention takes into account the linkage distance Dist (base coordinate distance) less than 100, R 2 SNP sites with a probability of >= 0.3. The maximum or optimal frequency combination of AB linkage inheritance (the final combination of all sites with the highest overall genetic probability) was identified as the reference genome composition of the Chinese population. The bases of the six points corresponding to the coordinates of the C2 maximum probability assembly sequence SEQ ID NO: 2 were modified. The coordinate points were modified with 0 as the starting coordinate to 1234: C, 1781: T, 997: G, 2220: C, 53: T, 2724: G, and the Gap region was removed. Although there were sites in linkage disequilibrium in the B2 population, the B2 maximum probability assembly genotype SEQ ID NO: 1 already met the high probability linkage inheritance pattern. Therefore, only the Gap region of the B2 maximum probability assembly genotype SEQ ID NO: 1 was removed. This resulted in the Chinese population B2 reference HBV genome sequence SEQ ID NO: 3 and the Chinese population C2 reference HBV genome sequence SEQ ID NO: 4.

[0046] Table 1: Linked genetic loci in the C2 population

[0047]

[0048] Table 2: Linked genetic loci in the B2 population

[0049]

[0050] Example 4: Common HBV mutations in the Chinese population and the B2 and C2 reference genomes in the Chinese population

[0051] The present invention collated common HBV variant sites and compared several large-scale genomic variation data of HBV strains in the Chinese population, including strain variation data from 1,301 Chinese chronic hepatitis B patients, strain variation data from 793 Chinese chronic hepatitis B patients, and 466 Chinese patients with liver cancer and hepatitis B. The corresponding sites in SEQ ID NO: 3 and SEQ ID NO: 4 were compared for consistency, as shown in Table 3 below. The results showed that the B2 and C2 reference genomes obtained in the present invention can well characterize the common mutation sites of HBV strains in Chinese chronic hepatitis B patients.

[0052] Table 3: Comparison of HBV common mutations and HBV mutation frequencies in the Chinese population and reference sequences

[0053]

[0054] Example 5: Construction of expression plasmids for the Chinese population B2 and C2 reference genome sequences

[0055] Literature research revealed that after one copy of HBV is inserted into a plasmid sequence, the corresponding transfected cells express low or no HBV-related proteins. Currently, the length of the inserted HBV sequence commonly used on the market is 1.1 / 1.3 times the original sequence. Some lengths can normally express HBV-related proteins after insertion, while others cannot. This is related to the characteristics of the HBV genome sequence. The complete HBV genome is an incomplete dsDNA. Before HBV is inserted into the plasmid, its circular genome needs to be linearized (currently, the starting coordinate position of the linear reference genome for circular DNA is often the EcoR1 restriction site. This site coordinate is defined as 1, but this does not mean that it is the breakpoint position). Therefore, the location of the HBV dsDNA breakpoint and the corresponding sequence position before transcription initiation are very important. The X gene region contains a promoter and enhancer. The present invention defines the starting position of the linear genome at coordinate 1031 (about 300bp before the start of the X gene CDS), and the genome ends at coordinate 1993 after one circle (about 150bp after the end sequence of the X gene CDS). 1.3 copies of the B2 and C2 reference genome sequences were obtained. HindIII / XhoI restriction sites were designed at both ends of the 1.3 copies of the B2 and C2 linear reference genomes and finally inserted into the corresponding restriction sites of pcDNA3.1(+), forming two plasmids: B2-pcDNA3.1 (nucleic acid sequence shown in SEQ ID NO: 5) and C2-pcDNA3.1 (nucleic acid sequence shown in SEQ ID NO: 6).

[0056] After the construction was completed, the inserted fragment was sequenced by first-generation sequencing to confirm that the sequencing sequence was consistent with the expectation, and the plasmid was extracted and endotoxin-free.

[0057] The sequence information of the vector sequencing primers involved in the sequencing is as follows:

[0058] 18T-seq-F:GATGTGCTGCAAGGCGATTAAG

[0059] 18T-seq-R:CTTTATGCTTCCGGCTCGTATG

[0060] Figure 3 、 Figure 4 It is the first generation sequencing result of the B2 and C2 reference HBV genome sequences in the plasmid.

[0061] Example 6: Construction and validation of a HBV drug screening model representative of the Chinese population

[0062] In the present invention, the HBV reference genome sequence was constructed entirely from scratch based on HBV sequence data infected with the Chinese population (>90%) and a very small number of East Asians. In order to verify the rationality of the constructed HBV B2 and C2 reference plasmids and the entire design process, the present invention selected commonly used HepG2 cells as the host cells for the two plasmids, and constructed in vitro screening cells B2-HepG2 and C2-HepG2 by transfecting plasmids B2-pcDNA3.1 and C2-pcDNA3.1 into HepG2 cells. The expression verification model was also verified by detecting intracellular HBV DNA, supernatant HBeAg, and supernatant HBsAg.

[0063] HBsAg is the main protein that HBV invades into human liver parenchymal cells. Serum HBsAg positivity is one of the markers of HBV infection. Therefore, the present invention focuses on the expression index of HBsAg.

[0064] The present invention selected HepG2 cells (ATCC number: HB-8065) ​​as the host cells for the B2-pcDNA3.1 and C2-pcDNA3.1 reference plasmids for experimental verification. The positive reference plasmid (abbreviated as D plasmid) was from WuXi AppTec. The plasmid vector was still pcDNA3.1(+). The HBV genome in the plasmid was the D type strain isolated from human hepatoblastoma (GenBank number U95551.1), which is a common reference sequence for HBV in in vitro screening models. The specific experimental methods and reagents used are as follows:

[0065] Unless the sources of reagents are specifically mentioned herein, these reagents can be obtained from any molecular biology reagent supplier under their trade names.

[0066] On day 0, HepG2 cells were transfected with B2-pcDNA3.1, C2-pcDNA3.1, and a positive reference plasmid as follows: HepG2 cells were washed with DPBS, digested with 0.05% trypsin, and gently pipetted in RPMI 1640 containing 2% FBS until single cells were counted. HBV plasmid DNA was then transferred into HepG2 cells using Fugene HD. Transfected cells were seeded at a density of 22,500 cells per well in a 96-well assay plate, with six replicates for each plasmid. Untransfected cells served as a blank control.

[0067] On the third day, cells and supernatant were collected and the intracellular HBV DNA and supernatant HBsAg and HBeAg were detected, respectively.

[0068] The qPCR method for detecting HBV DNA is briefly described as follows: NP-40 is added to each well to lyse cells, and the cell lysate is collected. The cell lysate is treated with DNase. After heat-inactivating the DNase, the lysate is transferred to a 96-well plate. After adding DNA extraction reagent, the plate is heated at 65°C for 6 minutes and then at 98°C for 2 minutes to lyse the virus. HBV DNA in the sample is quantified by qPCR. The reaction system is shown in Table 5. HBV plasmid DNA is used as a standard and serially diluted 10-fold, with the standard ranging from 1.0 × 10 7 copies / μl to 10 copies / μl. The sequence information of the corresponding primers and probes is as follows:

[0069] Forward primer sequence: CGTCTGTGCCTTCTCATCT

[0070] Reverse primer sequence: GTTCACGGTGGTCTCCAT

[0071] Probe sequence: ACTTCGCTTCACCTCTGCACGTC

[0072] Table 4: qPCR reaction system

[0073] Reagents Volume required to prepare 1 well (μl) Quantitative Rapid Start Universal Probe Reagent 5 HBV forward primer (10 μM) 0.4 HBV reverse primer (10 μM) 0.4 HBV probe (10 μM) 0.2 AE buffer (DNA elution buffer) 2 DNA or standards 2

[0074] The PCR reaction program was: 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles. The HBV DNA content in the samples was calculated based on the standard curve and the Ct value of each sample.

[0075] The ELISA test refers to the instructions of the Antu Biotech kit (Cat. No.: Antu Biotech-CL 0310). The method is briefly described as follows: the kit is equilibrated at room temperature for 1 hour, 50 μl of sample is added to each well, and then 50 μl of enzyme conjugate is added and incubated at 37°C for 60 minutes. Wash the plate 6 times, add 50 μl of luminescent substrate, and incubate at room temperature in the dark for 10 minutes. Finally, the luminescence intensity is detected. The HBeAg and HBsAg standards range from 0.1-200 PEIU / ml and 0.05-250 IU / ml, respectively. The standard curve HBeAg constructed by the experimental test is shown in the figure. Figure 5 , HBsAg diagram Figure 6 .

[0076] The experimental results demonstrate the success of the in vitro screening model constructed using the present invention's process, with all corresponding HBV components within the effective detection range (see Tables 5 and 6). HBV DNA was highly expressed in cells. HBsAg expression levels for the B2-pcDNA3.1 and C2-pcDNA3.1 reference plasmids were higher than those for the D reference plasmid. HBeAg expression levels for the C2-pcDNA3.1 reference plasmid were comparable to those for the D reference plasmid. HBeAg expression levels for the B2-pcDNA3.1 reference plasmid were within the effective detection range.

[0077] Table 5: Expression effects of HBV DNA in different plasmids

[0078]

[0079] Table 6: Expression effects of HBeAg and HBsAg from different plasmids

[0080]

[0081] Example 6: Screening dsRNA molecules using a representative in vitro anti-HBV screening system for the Chinese population

[0082] To evaluate the suitability of the in vitro screening system constructed in the present invention for screening siRNA compounds, two pairs of dsRNA compounds plus a positive siRNA control were synthesized in this example to evaluate the inhibitory effects of the test compounds on HBV. The positive control was a compound (vir-2218) disclosed in US Pat. No. 11492623B2, which has completed a Phase II clinical trial in China for the treatment of chronic hepatitis B, with the corresponding experimental number NCT04507269. The 2-19 nt sequence of the SS strand of the test compound BPR301-500101 completely matched the B2 and C2 sequences constructed in the present invention; the SS strand sequence of the test compound BPR301-300701 completely matched the B2 and C2 sequences constructed in the present invention. siRNAs matching the B2 and C2 sequences of the present invention demonstrated a strong inhibitory effect in the model, demonstrating the broad applicability of the in vitro anti-HBV screening system developed in this invention for screening dsRNA molecules representative of the Chinese population.

[0083] 1. Synthesis of dsRNA molecules

[0084] The specific sequences and modifications of dsRNA involved in the experiment are shown in Table 7, and the modifications of different nucleotide monomers are shown in Table 8. In the case where the source of reagents is not specifically given in this article, these reagents can be obtained from any molecular biology reagent supplier with the quality / purity standards used in molecular biology.

[0085] The siRNA sense and antisense strand sequences were synthesized on a 48-channel nucleic acid synthesizer (LK-48E) using a solid support-mediated phosphoramidite chemistry method at a 1 μmol scale. The solid support was loaded with a universal solid support (Universal). Auxiliary synthesis reagents, 2′-F and 2′-O methyl RNA and deoxyphosphoramidites were obtained from Wuhu Huaren. The corresponding phosphoramidites were used to introduce 2′-F 2′-O-methyl, and the synthesis of 3′-conjugated single strands was performed on a solid CPG support. The CPG universal solid support was used to synthesize the antisense single strand. 5-Ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M in acetonitrile), and the coupling time for all phosphoramidites (0.1 M in acetonitrile) was 3 minutes. Phosphorothioate linkages were generated using 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, 0.1 M solution in anhydrous acetonitrile / pyridine (1:1 v / v). Oxidation time was 3 minutes. All sequences were synthesized with the DMT group finally removed ("DMT-free (DMT-free)"). After solid-phase synthesis, the oligonucleotides were cleaved from the solid support and aminolyzed in a sealed centrifuge tube at 55°C for 5 hours using 300 μL of concentrated ammonia solution. At the end of the cleavage and deprotection steps, the centrifuge tubes were allowed to reach room temperature, the solid support was removed by filtration, and the mother liquor was concentrated to dryness on a centrifugal concentrator. After concentration, 200 μL of pure water was added for reconstitution, and samples were collected and sent for testing. Samples from each sequence were analyzed by LC-MS to confirm molecular weight and purity. The crude product was separated and purified by HPLC using IRRP with 100 mM triethylamine acetate and acetonitrile as the mobile phase. Samples were collected and concentrated to dryness on a centrifugal concentrator. Purity was determined by LC-MS, and samples that met the purity requirements were used for annealing. An equimolar mixture of sense and antisense single strands was combined in a centrifuge tube and annealed. After combining the complementary single strands, the centrifuge tubes were heated to 55°C in a water bath for 20-30 minutes, then removed and allowed to stand at room temperature. Nanodrop The concentration was tested on one, and the samples were divided into 0.5 ml centrifuge tubes as required, and the divided samples were freeze-dried.

[0086] Table 7: Modified nucleotide sequences of HBV dsRNA agents

[0087]

[0088] Table 8: Abbreviations of nucleotide monomers used in the representation of modified nucleic acid sequences

[0089]

[0090]

[0091] 2. In vitro screening of dsRNA molecules

[0092] The present invention uses the two anti-HBV in vitro screening models with infection characteristics of the Chinese population constructed in this paper to screen compounds. For specific test information, please refer to Table 9. All the tested compounds have different degrees of inhibitory effects on HBV RNA and HBsAg in the two in vitro screening models. The results are shown in Table 9. Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 The results showed that the constructed B2-HepG2 and C2-HepG2 in vitro screening cells can be used for in vitro screening of nucleic acid drugs.

[0093] Table 9: In vitro models and screening concentrations of the tested compounds

[0094] Compound In vitro screening models Screening concentration (nM) Detection indicators BPR301-300701 B2-HepG2 1、0.5、0.1 HBV RNA, HBsAg BPR301-500101 B2-HepG2 1、0.5、0.1 HBV RNA, HBsAg vir-2218 B2-HepG2 1、0.5、0.1 HBV RNA, HBsAg BPR301-300701 C2-HepG2 1、0.5、0.1 HBV RNA, HBsAg BPR301-500101 C2-HepG2 1、0.5、0.1 HBV RNA, HBsAg vir-2218 C2-HepG2 1、0.5、0.1 HBV RNA, HBsAg

[0095] The specific experimental methods and reagents used are as follows:

[0096] Unless the sources of reagents are specifically mentioned herein, these reagents can be obtained from any molecular biology reagent supplier under their trade names.

[0097] On day 0, HepG2 cells were transfected with the tested HBV plasmid DNA, and the transfected cells were seeded into 96-well test plates at a density of 22,500 cells per well.

[0098] On day 1, cells were transfected with test compounds using RNAiMax. The control compound, vir-2218, started at a 10 nM concentration and was diluted to 1 nM, 0.5 nM, and 0.1 nM. PBS was used as a negative control. Each concentration was replicated in triplicate. Cells were cultured at 37°C with 5% CO₂ for 3 days, followed by replacement with fresh medium without compound for an additional 3 days.

[0099] On day 6, cell supernatants were collected for HBsAg assay, and cells were harvested for RNA extraction. RT-PCR was performed to detect target gene mRNA in the samples. GAPDH was also detected as an internal reference gene. Intracellular RNA was extracted using the RNeasy kit (QIAGEN-74182) according to the manufacturer's instructions. RNA was then reverse-transcribed into cDNA using the FastKing RT Kit (with gDNase) (TIANGEN-KR116). HBV mRNA expression was then measured using real-time fluorescence quantitative PCR.

[0100] The relative expression level of the target gene was calculated using 2 -ΔΔCT The calculation formula is as follows:

[0101] ΔCT = average Ct value of target gene - average Ct value of reference gene;

[0102] ΔΔCT = ΔCT (drug-treated group) - ΔCT (control group);

[0103] mRNA relative expression = 2 -ΔΔCT

[0104] Data Analysis:

[0105] % HBV RNA inhibition rate = (1-test sample HBV RNA level / control PBS HBV RNA level) × 100%

[0106] HBV surface antigen was detected using an ELISA method, as described in the instructions for the Antu Biotech kit (Cat. No.: Antu Biotech-CL0310). The method is briefly described as follows: The kit was equilibrated at room temperature for 1 hour. 50 μl of sample was added to each well, followed by 50 μl of enzyme conjugate and incubation at 37°C for 60 minutes. The plate was washed six times, and 50 μl of luminescent substrate was added and incubated at room temperature in the dark for 10 minutes. Finally, the luminescence intensity was measured.

[0107] Seq ID NO:1:B2 Maximum probability sequence

[0108] CTCCACCACTTTCCACCAAACTCTTCAAGATCCCAGAGTCAGGGCCCTGTACTTTCCTGCTGGTGG

[0109] CTCCAGTCAGGAACAGTGAGCCCTGCTCAGAATACTGTCTCTGCCATATCGTCAATCTTATCGAAG

[0110] ACTGGGGACCCTGTACCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTA

[0111] CAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACT

[0112] TCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTC

[0113] ACTCACCAACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATC

[0114] TTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCC

[0115] GTTTGTCCTCTAATTCCAGGATCATCAACAACCAGCACCGGACCATGCAAAACCTGCACAACTCCT

[0116] GCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTA

[0117] TTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTG

[0118] GCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGTTA

[0119] TATGGATGATGTGGTTTTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTATGCCGCTGTTACC

[0120] AATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTCACAAAACAAAAAGATGGGGATATTCCCTTAA

[0121] CTTCATGGGATATGTAATTGGGAGTTGGGGCACATTGCCACAGGAACATATTGTACAAAAAATCAAA

[0122] ATGTGTTTTAGGAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTC

[0123] TTTTGGGGTTTGCCGCCCCTTTCACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATAC

[0124] AAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTGAACC

[0125] TTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTT

[0126] GGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGC

[0127] GGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGGGCAAAACTCATCGGGACTGACAATTC

[0128] TGTCGTGCTCTCCCGCAAGTATACATCATTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTG

[0129] CGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGC

[0130] TTGGGGCTCTACCGCCCGCTTCTCCGCCTGTTGTACCGACCGACCACGGGGCGCACCTCTCTTTAC

[0131] GCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCG

[0132] CATGGAGACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGAC

[0133] TTTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTGTGTTTACTGAGTGGGAGG

[0134] AGTTGGGGGAGGAGGTTAGGTTAAAGGTCNNNTTTGTACTAGGAGGCTGTAGGCATAAATTGGTGT

[0135] GTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTCATGTCCTACTGTTCAAG

[0136] CCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTATAAAGAATTTGGAGCTT

[0137] CTGTGGAGTTACTCTCNNNNNNTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTCGAC

[0138] ACCGCCTCTGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACGGCAC

[0139] TCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTAGCCACCTGGGTGGGAAGTAATTTGG

[0140] AAGATCCAGCATCCAGGGAATTAGTAGTCAGCTATGTCAACGTTAATATGGGCCTAAAAATCAGACA

[0141] ACTATTGTGGTTTCACATTTCCTGTCTTACTTTTGGGAGAGAAACTGTTCTTGAATATTTGGTGTCTT

[0142] TTGGAGTGTGGATTCGCACTCCTCCTGCATATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG

[0143] GAAACTACTGTTGTTAGANNNNNNCGAAGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCG

[0144] CAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAGTATTCC

[0145] TTGGACACATAAGGTGGGAAACTTTACGGGGCTTTATTCTTCTACGGTACCTTGCTTTAATCCTAAAT

[0146] GGCAAACTCCTTCTTTTCCTGACATTCATTTGCAGGAGGACATTGTTGATAGATGTAAGCAATTTGT

[0147] GGGGCCCCTTACAGTAAATGAAAACAGGAGACTAAAATTAATTATGCCTGCTAGGTTTTATCCCAAT

[0148] GTTACTAAATATTTGCCCTTAGATAAAGGGATCAAACCGTATTATCCAGAGTATGTAGTTAATCATTAC

[0149] TTCCAGACGCGACATTATTTACACACTCTTTGGAAGGCGGGGATCTTATATAAAAGAGAGTCCACAC

[0150] GTAGCGCCTCATTTTGCGGGTCACCATATTCTTGGGAACAAGATCTACAGCATGGGAGGTTGGTCTT

[0151] CCAAACCTCGAAAAGGCATGGGGACAAATCTTTCTGTCCCCAATCCCCTGGGATTCTTCCCCGATCA

[0152] TCAGTTGGACCCTGCATTCAAAGCCAACTCAGAAAATCCAGATTGGGACCTCAACCCGCACAAGG

[0153] ACAACTGGCCGGACGCCAACAAGGTGGGAGTGGGAGCATTCGGGCCAGGGTTCACCCCTCCCCAT

[0154] GGGGGACTGTTGGGGTGGAGCCCTCAGGCTCAGGGCCTACTCACAACTGTGCCAGCAGCTCCTCC

[0155] TCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCTACTCCCTTATCTCCACCTCTAAGGGACAC

[0156] TCATCCTNNNNNNCAGGCCATGNNNCAGTGGAA

[0157] Seq ID NO:2: C2 Most Probable Sequence

[0158] NNCTCCACAACATTCCACCAAGCTCTGCTAGATCCCAGAGTGAGGGGCCTATACTTTCCTGCTGGT

[0159] GGCTCCAGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCACCCATATCGTCAATCTTCTCGA

[0160] GGACTGGGGACCCTGCACCGAACATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTG

[0161] TTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGG

[0162] ACTTCTCTCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCC

[0163] AATCACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTAT

[0164] CATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTT

[0165] GCCCGTTTGTCCTCTACTTCCAGGAANNNCATCAACTACCAGCACGGGACCATGCAAGACCTGCAC

[0166] GATTCCTGCTCAAGGAACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGC

[0167] ACTTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTT

[0168] CTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTT

[0169] CAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTA

[0170] TTACCAATTTTCTTTTGTCTTTGGGTATACATTTGAACCCTAATAAAACCAAACGTTGGGGCTACTCC

[0171] CTTAACTTCATGGGATATGTAATTGGAAGTTGGGGTACTTTACCGCAAGAACATATTGTACTAAAAAT

[0172] CAAGCAATGTTTTCGAAAACTGCCTGTAAATAGACCTATTGATTGGAAAGTATGTCAAAGAATTGTG

[0173] GGTCTTTTGGGCTTTGCTGCCCCTTTTACACAATGTGGCTATCCTGCCTTAATGCCTTTATATGCATGT

[0174] ATACAATCTAAGCAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTGTGTAAACAATATCTGA

[0175] ACCTTTACCCCGTTGCCCGGCAACGGTCAGGTCTCTGCCAAGTGTTTGCTGACGCAACCCCCACTG

[0176] GATGGGGCTTGGCTATTGGCCATCGGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATAC

[0177] TGCGGAACTCCTAGCAGCTTGTTTTGCTCGCAGCCGGTCTGGAGCGAAACTTATCGGAACCGACAA

[0178] CTCTGTTGTCCTCTCTCGGAAATACACCTCCTTTCCATGGCTGCTAGGGTGTGCTGCCAACTGGATC

[0179] CTGCGCGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCGTCTCGGGGC

[0180] CGTTTGGGACTCTACCGTCCCCTTCTTCATCTGCCGTTCCGGCCGACCACGGGGCGCACCTCTCTTT

[0181] ACGCGGTCTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGT

[0182] CGCATGGAGACCACCGTGAACGCCCACCANNNNNNNNNNNNNNNGGTCTTGCCCAAGGTCTTACA

[0183] TAAGAGGACTCTTGGACTCTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTTT

[0184] GTTTAAGGACTGGGAGGAGTTGGGGGAGGAGATTAGGTTAAAGATCTTTGTACTAGGAGGCTGTAG

[0185] GCATAAATTGGTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTCATG

[0186] TCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTATAAA

[0187] GAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCT

[0188] CCTCGACACCGCCTCTGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCAT

[0189] ACAGCACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTGGCCACCTGGGTGGGAAGT

[0190] AATTTGGAAGACCCAGCATCCAGGGAATTAGTAGTCAGCTATGTCAATGTTAATATGGGCCTAAAAA

[0191] TCAGACAACTATTGTGGTTTCACATTTCCTGTCTTACTTTTGGAAGAGAAACTGTTCTTGAGTATTTG

[0192] GTGTCTTTTGGAGTGTGGATTCGCACTCCTCCTGCTTACAGACCACCAAATGCCCCTATCTTATCAA

[0193] CACTTCCGGAAACTACTGTTGTNNNNNNTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCC

[0194] TCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGT

[0195] TAGTATCCCTTGGACTCATAAGGTGGGAAACTTTACTGGGCTTTATTCTTCTACTGTACCTGTCTTTA

[0196] ATCCTGAGTGGCAAACTCCCTCCTTTCCTCACATTCATTTACAGGAGGACATTATTAATAGATGTCAA

[0197] CAATATGTGGGCCCTCTTACAGTTAATGAAAAAAGGAGATTAAAATTAATTATGCCTGCTAGGTTCTA

[0198] TCCTAACCTTACCAAATATTTGCCCTTGGACAAAGGCATTAAACCATATTATCCTGAACATGCAGTTA

[0199] ATCATTACTTCAAAACTAGGCATTATTTACATACTCTGTGGAAGGCTGGCATTCTATATAAGAGAGAA

[0200] ACTACACGCAGCGCCTCATTTTGTGGGTCACCATATTCTTGGGAACAAGAGCTACAGCATGGGAGG

[0201] TTGGTCTTCCAAACCTCGACAAGGCATGGGGACGAATCTTTCTGTTCCCAATCCTCTGGGATTCTTT

[0202] CCCGATCACCAGTTGGACCCTGCGTTCGGAGCCAACTCAAACAATCCAGATTGGGACTTCAACCCC

[0203] AACAAGGATCACTGGCCAGAGGCAAATCAGGTAGGAGCGGGAGCATTCGGGCCAGGGTTCACCCC

[0204] ACCACACGGCGGTCTTTTGGGGTGGAGCCCTCAGGCTCAGGGCATATTGACAACAGTGCCAGCAG

[0205] CACCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGACAGCCTACTCCCATCTCTCCACCTCTAA

[0206] GAGACAGTCATCCTCAGGCCATGCAGTGGAA

[0207] Seq ID NO:3B2 Final Sequence

[0208] CTCCACCACTTTCCACCAAACTCTTCAAGATCCCAGAGTCAGGGCCCTGTACTTTCCTGCTGGTGG

[0209] CTCCAGTTCAGGAACAGTGAGCCCTGCTCAGAATACTGTCTCTGCCATATCGTCAATCTTATCGAAG

[0210] ACTGGGGACCCTGTACCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTA

[0211] CAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACT

[0212] TCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTC

[0213] ACTCACCAACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATC

[0214] TTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCC

[0215] GTTTGTCCTCTAATTCCAGGATCATCAACAACCAGCACCGGACCATGCAAAACCTGCACAACTCCT

[0216] GCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGTA

[0217] TTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTG

[0218] GCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGTTA

[0219] TATGGATGATGTGGTTTTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTATGCCGCTGTTACC

[0220] AATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTCACAAAACAAAAAGATGGGGATATTCCCTTAA

[0221] CTTCATGGGATATGTAATTGGGAGTTGGGGCACATTGCCACAGGAACATATTGTACAAAAAATCAAA

[0222] ATGTGTTTTAGGAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTC

[0223] TTTTGGGGTTTGCCGCCCCTTTCACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATAC

[0224] AAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTGAACC

[0225] TTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTT

[0226] GGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGC

[0227] GGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGGGCAAAACTCATCGGGACTGACAATTC

[0228] TGTCGTGCTCTCCCGCAAGTATACATCATTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTG

[0229] CGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGC

[0230] TTGGGGCTCTACCGCCCGCTTCTCCGCCTGTTGTACCGACCGACCACGGGGCGCACCTCTCTTTAC

[0231] GCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCG

[0232] CATGGAGACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGAC

[0233] TTTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTGTGTTTACTGAGTGGGAGG

[0234] AGTTGGGGGAGGAGGTTAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTGTGTT

[0235] CACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTCATGTCCTACTGTTCAAGCCT

[0236] CCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTG

[0237] TGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTCGACACCGCCTCT

[0238] GCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACGGCACTCAGGCAA

[0239] GCTATTCTGTGTTGGGGTGAGTTGATGAATCTAGCCACCTGGGTGGGAAGTAATTTGGAAGATCCA

[0240] GCATCCAGGGAATTAGTAGTCAGCTATGTCAACGTTAATATGGGCCTAAAAATCAGACAACTATTGT

[0241] GGTTTCACATTTCCTGTCTTACTTTTGGGAGAGAAACTGTTCTTGAATATTTGGTGTCTTTTGGAGTG

[0242] TGGATTCGCACTCCTCCTGCATATAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTA

[0243] CTGTTGTTAGACGAAGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCT

[0244] CAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAGTATTCCTTGGACACATAAG

[0245] GTGGGAAACTTTACGGGGCTTTATTCTTCTACGGTACCTTGCTTTAATCCTAAATGGCAAACTCCTTC

[0246] TTTTCCTGACATTCATTTGCAGGAGGACATTGTTGATAGATGTAAGCAATTTGTGGGGCCCCTTACA

[0247] GTAAATGAAAACAGGAGACTAAAATTAATTATGCCTGCTAGGTTTTATCCCAATGTTACTAAATATTT

[0248] GCCCTTAGATAAAGGGATCAAACCGTATTATCCAGAGTATGTAGTTAATCATTACTTCCAGACGCGA

[0249] CATTATTTACACACTCTTTGGAAGGCGGGGATCTTATATAAAAGAGAGTCCACACGTAGCGCCTCAT

[0250] TTTGCGGGTCACCATATTCTTGGGAACAAGATCTACAGCATGGGAGGTTGGTCTTCCAAACCTCGA

[0251] AAAGGCATGGGGACAAATCTTTCTGTCCCCAATCCCCTGGGATTCTTCCCCGATCATCAGTTGGACC

[0252] CTGCATTCAAAGCCAACTCAGAAAATCCAGATTGGGACCTCAACCCGCACAAGGACAACTGGCCG

[0253] GACGCCAACAAGGTGGGAGTGGGAGCATTCGGGCCAGGGTTCACCCCTCCCCATGGGGGACTGTT

[0254] GGGGTGGAGCCCTCAGGCTCAGGGCCTACTCACAACTGTGCCAGCAGCTCCTCCTCCTGCCTCCAC

[0255] CAATCGGCAGTCAGGAAGGCAGCCTACTCCCTTATCTCCACCTCTAAGGGACACTCATCCTCAGGC

[0256] CATGCAGTGGAA

[0257] Seq ID NO:4C2 Final Sequence

[0258] CTCCACAACATTCCACCAAGCTCTGCTAGATCCCAGAGTGAGGGGCCTATATTTTCCTGCTGGTGGC

[0259] TCCAGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCACCCATATCGTCAATCTTCTCGAGGA

[0260] CTGGGGACCCTGCACCGAACATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTAC

[0261] AGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTT

[0262] CTCTCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATC

[0263] ACTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATA

[0264] TTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCC

[0265] CGTTTGTCCTCTACTTCCAGGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATTCC

[0266] TGCTCAAGGAACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGT

[0267] ATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTG

[0268] GCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTA

[0269] TATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCA

[0270] ATTTTCTTTTGTCTTTGGGTATACATTTGAACCCTAATAAAACCAAACGTTGGGGCTACTCCCTTAAC

[0271] TTCATGGGATATGTAATTGGAAGTTGGGGTACTTTACCGCAAGAACATATTGTACTAAAAATCAAGC

[0272] AATGTTTTCGAAAACTGCCTGTAAATAGACCTATTGATTGGAAAGTATGTCAGAGAATTGTGGGTCT

[0273] TTTGGGCTTTGCTGCCCCTTTTACACAATGTGGCTATCCTGCCTTAATGCCTTTATATGCATGTATACA

[0274] ATCTAAGCAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTGTGTAAACAATATCTGAACCTT

[0275] TACCCCGTTGCCCGGCAACGGTCAGGTCTCTGCCAAGTGTTTGCTGACGCAACCCCCACTGGATGG

[0276] GGCTTGGCTATTGGCCATCGCCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGG

[0277] AACTCCTAGCAGCTTGTTTTGCTCGCAGCCGGTCTGGAGCGAAACTTATCGGAACCGACAACTCTG

[0278] TTGTCCTCTCTCGGAAATACACCTCCTTTCCATGGCTGCTAGGGTGTGCTGCCAACTGGATCCTGCG

[0279] CGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCGTCTCGGGGCCGTTT

[0280] GGGACTCTACCGTCCCCTTCTTCATCTGCCGTTCCGGCCGACCACGGGGCGCACCTCTCTTTACGCG

[0281] GTCTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCAT

[0282] GGAGACCACCGTGAACGCCCACCAGGTCTTGCCCAAGGTCTTACATAAGAGGACTCTTGGACTCTC

[0283] AGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTTTGTTTAAGGACTGGGAGGAGTT

[0284] GGGGGAGGAGATTAGGTTAATGATCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTCTGTTCACCA

[0285] GCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTCATGTCCTACTGTTCAAGCCTCCAAG

[0286] CTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTGTGGAG

[0287] TTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTCGACACCGCCTCTGCTCTG

[0288] TATCGGGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACAGCACTCAGGCAAGCTATTC

[0289] TGTGTTGGGGTGAGTTGATGAATCTGGCCACCTGGGTGGGAAGTAATTTGGAAGACCCAGCATCCA

[0290] GGGAATTAGTAGTCAGCTATGTCAATGTTAATATGGGCCTAAAAATCAGACAACTACTGTGGTTTCA

[0291] CATTTCCTGTCTTACTTTTGGAAGAGAAACTGTTCTTGAGTATTTGGTGTCTTTTGGAGTGTGGATTC

[0292] GCACTCCTCCTGCTTACAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGT

[0293] TAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGC

[0294] CGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAGTATCCCTTGGACTCATAAGGTGGGAA

[0295] ACTTTACTGGGCTTTATTCTTCTACTGTACCTGTCTTTAATCCTGAGTGGCAAACTCCCTCCTTTCCT

[0296] CACATTCATTTACAGGAGGACATTTAATAGATGTCAACAATATGTGGGCCCTCTTACAGTTAATGA

[0297] AAAAGGAGATTAAATTTATGCCTGCTAGGTTCTATCCTAACCTTACCAAATTTGCCCTTGG

[0298] ACAAAGGCATTAAACCGTTATCCTGAACATGCAGTTAATCATTACTTCAAAACTAGGCATTATTTA

[0299] CATACTCTGTGGAAGGCTGGCATTCTATATAAGAGAGAAACTACACGCAGCGCCTCATTTTGTGGGT

[0300] CACCATATTCTTGGGAACAAGAGCTACAGCATGGGAGGTTGGTCTTCCAAACCTCGACAAGGCATG

[0301] GGGACGAATCTTTCTGTTCCCAATCCTCTGGGATTCTTTCCCGATCACCAGTTGGACCCTGCGTTCG

[0302] GAGCCAACTCAAACAATCCAGATTGGGACTTCAACCCCAACAAGGATCACTGGCCAGAGGCAAAT

[0303] CAGGTAGGAGCGGGAGCATTCGGGCCAGGGTTCACCCCACCACAGGCGGTCTTTTGGGGTGGAG

[0304] CCCTCAGGCTCAGGGCATATTGACAACAGGTGCCAGCAGCACCTCCTCCTGCCTCCACCAATCGGCA

[0305] GTCAGGAAGACAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTG

[0306] GAA

[0307] Seq ID NO:5 B2-pcDNA3.1

[0308] TGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG

[0309] TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG

[0310] CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAA

[0311] GGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC

[0312] TTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA

[0313] TAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG

[0314] GGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG

[0315] CACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGC

[0316] ACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGG

[0317] AGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGC

[0318] ATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTT

[0319] GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG

[0320] AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT

[0321] TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT

[0322] GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT

[0323] ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTC

[0324] CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATC

[0325] AATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGG

[0326] AGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

[0327] CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAA

[0328] CCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTT

[0329] TAAACTTAAGCTTCGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACA

[0330] GGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTGAACCTTTACCCCGTT

[0331] GCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGC

[0332] CATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTA

[0333] GCCGCTTGTTTTGCTCGCAGCAGGTCTGGGGCAAAACTCATCGGGACTGACAATTCTGTCGTGCTC

[0334] TCCCGCAAGTATACATCATTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGT

[0335] CCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCT

[0336] ACCGCCCGCTTCTCCGCCTGTTGTACCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCC

[0337] CGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACC

[0338] ACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATG

[0339] TCAACGACCGACCTTGAGGCATACTTCAAAGACTGTGTGTTTACTGAGTGGGAGGAGTTGGGGGA

[0340] GGAGGTTAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTGTGTTCACCAGCACC

[0341] ATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTG

[0342] CCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTGTGGAGTTACTC

[0343] TCTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTCGACACCGCCTCTGCTCTGTATCG

[0344] GGAGGCCTTAGAGTCTCCGGAACATTGTTCACCTCACCATACGGCACTCAGGCAAGCTATTCTGTG

[0345] TTGGGGTGAGTTGATGAATCTAGCCACCTGGGTGGGAAGTAATTTGGAAGATCCAGCATCCAGGGA

[0346] ATTAGTAGTCAGCTATGTCAACGTTAATATGGGCCTAAAAATCAGACAACTATTGTGGTTTCACATTT

[0347] CCTGTCTTACTTTTGGGAGAGAAACTGTTCTTGAATATTTGGTGTCTTTTGGAGTGTGGATTCGCAC

[0348] TCCTCCTGCATATAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGAC

[0349] GAAGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGT

[0350] CGCAGAAGATCTCAATCTCGGGAATCTCAATGTTTAGTATTCCTTGGACACATAAGGTGGGAAACTTT

[0351] ACGGGGCTTTATTCTTCTACGGTACCTTGCTTTAATCCTAAATGGCAAACTCCTTCTTTTCCTGACAT

[0352] TCATTTGCAGGAGGACATTGTTGATAGATGTAAGCAATTTGTGGGGCCCCTTCAGTAAATGAAAAC

[0353] AGGAGACTAAAATTAATTATGCCTGCTAGGTTTTATCCCAATGTTACTAAATATTTGCCCTTAGATAA

[0354] AGGGATCAAACCGATTATCCAGAGTATGTAGTTAATCATTACTTCCAGACGCGACATTATTTACACA

[0355] CTCTTTGGAAGGCGGGATCTTATATAAAAGAGAGTCCACACGTAGCGCCTCATTTTGCGGGTCAC

[0356] CATATTCTTGGGAACAAGATCTACAGCATGGGAGGTTGGTCTTCCAAACCTCGAAAAGGCATGGGG

[0357] ACAAATCTTTCTGTCCCCAATCCCCTGGGATTCTTCCCCGATCATCAGTTGGACCCTGCATTCAAAG

[0358] CCAACTCAGAAAATCCAGATTGGGACCTCCAACCCGCACAAGGACAACTGGCCGGACGCCAACAA

[0359] GGTGGGAGTGGGAGCATTCGGGCCAGGGTTCACCCCTCCCCATGGGGGACTGTTGGGGTGGAGCC

[0360] CTCAGGCTCAGGGCCTACTCACAACTGTGCCAGCAGCTCCTCCTCCTGCCTCCACCAATCGGCAGT

[0361] CAGGAAGGCAGCCTACTCCCTTATCTCCACCTCTAAGGGACACTCATCCTCAGGCCATGCAGTGGA

[0362] ACTCCACCACTTTCCACCAAACTCTTCAAGATCCCAGAGTCAGGGCCCTGTACTTTCCTGCTGGTG

[0363] GCTCCAGTTCAGGAACAGTGAGCCCTGCTCAGAATACTGTCTCTGCCATATCGTCAATCTTATCGAA

[0364] GACTGGGGACCCTGTACCGAACATGGAGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTT

[0365] ACAGGCGGGGTTTTTCTTGTTGACAAAAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGAC

[0366] TTCTCTCAATTTTCTAGGGGGAACACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGT

[0367] CACTCACCAACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCAT

[0368] CTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCC

[0369] CGTTTGTCCTCTAATTCCAGGATCATCAACAACCAGCACCGGACCATGCAAAACCTGCACAACTCC

[0370] TGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGACGGAAACTGCACCTGT

[0371] ATTCCCATCCCATCATCTTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTG

[0372] GCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTCTGGCTTTCAGTTA

[0373] TATGGATGATGTGGTTTTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTATGCCGCTGTTACC

[0374] AATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTCACAAAACAAAAAGATGGGGATATTCCCTTAA

[0375] CTTCATGGGATATGTAATTGGGAGTTGGGGCACATTGCCACAGGAACATATTGTACAAAAAATCAAA

[0376] ATGTGTTTTAGGAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTC

[0377] TTTTGGGGTTTGCCGCCCCTTTCACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATAC

[0378] AAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTGAACC

[0379] TTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTT

[0380] GGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGC

[0381] GGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGGGCAAAACTCATCGGGACTGACAATTC

[0382] TGTCGTGCTCTCCCGCAAGTATACATCATTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTG

[0383] CGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGC

[0384] TTGGGGCTCTACCGCCCGCTTCTCCGCCTGTTGTACCGACCGACCACGGGGCGCACCTCTCTTTAC

[0385] GCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCG

[0386] CATGGAGACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGAC

[0387] TTTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTGTGTTTACTGAGTGGGAGG

[0388] AGTTGGGGGAGGAGGTTAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAAATTGGTGTGTT

[0389] CACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTCATGTCCTACTGTTCAAGCCT

[0390] CCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTG

[0391] TGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTCCTCGAGTCTAGA

[0392] GGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC

[0393] CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGA

[0394] AATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAA

[0395] GGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGG

[0396] CGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCG

[0397] GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTC

[0398] GCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCC

[0399] CTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTC

[0400] ACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAAT

[0401] AGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAG

[0402] GGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTA

[0403] ATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATG

[0404] CAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAG

[0405] AAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCG

[0406] CCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGA

[0407] GGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGG

[0408] CTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGA

[0409] TCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTA

[0410] TTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCG

[0411] CAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGA

[0412] GGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCAC

[0413] TGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCT

[0414] TGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCT

[0415] ACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGG

[0416] TCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAG

[0417] GCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAA

[0418] TATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCG

[0419] CTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCG

[0420] CTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACG

[0421] AGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCAC

[0422] GAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCG

[0423] GCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGC

[0424] AGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGC

[0425] ATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCT

[0426] AGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACA

[0427] CAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATT

[0428] AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC

[0429] GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCG

[0430] CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCC

[0431] ACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC

[0432] GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC

[0433] GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA

[0434] AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTT

[0435] CGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC

[0436] CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG

[0437] TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG

[0438] CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG

[0439] AAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC

[0440] TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG

[0441] AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA

[0442] CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA

[0443] AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC

[0444] AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA

[0445] GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACG

[0446] CTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC

[0447] CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCC

[0448] AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT

[0449] ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA

[0450] AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCAT

[0451] GGTTATGGCAGCAC

[0452] Seq ID NO:6 C2-pcDNA3.1

[0453] TGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA

[0454] AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT

[0455] ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACT

[0456] CTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTC

[0457] AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAA

[0458] AGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC

[0459] ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATA

[0460] GGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGAT

[0461] CCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCT

[0462] GCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTT

[0463] GACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGG

[0464] CCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAG

[0465] TTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC

[0466] CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT

[0467] TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTAT

[0468] CATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCA

[0469] GTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT

[0470] GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA

[0471] GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAAT

[0472] GTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATA

[0473] AGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCA

[0474] CTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTCACAATGTGGCTATCCTGCCTTAA

[0475] TGCCTTTATATGCATGTATACAATCTAAGCAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCT

[0476] GTGTAAACAATATCTGAACCTTTACCCCGTTGCCCGGCAACGGTCAGGTCTCTGCCAAGTGTTTGC

[0477] TGACGCAACCCCCACTGGATGGGGCTTGGCTATTGGCCATCGCCGCATGCGTGGAACCTTTGTGG

[0478] CTCCTCTGCCGATCCATACTGCGGAACTCCTAGCAGCTTGTTTTGCTCGCAGCCGGTCTGGAGCGA

[0479] AACTTATCGGAACCGACAACTCTGTTGTCCTCTCTCGGAAATACACCTCCTTTCCATGGCTGCTAG

[0480] GGTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCCCG

[0481] CGGACGACCCGTCTCGGGGCCGTTTGGGACTCTACCGTCCCCTTCTTCATCTGCCGTTCCGGCCGA

[0482] CCACGGGGCGCACCTCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGC

[0483] ACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCAGGTCTTGCCCAAGG

[0484] TCTTACATAAGAGGACTCTTGGACTCTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAA

[0485] GACTGTTTGTTTAAGGACTGGGAGGAGTTGGGGGAGGAGATTAGGTTAATGATCTTTGTACTAGG

[0486] AGGCTGTAGGCATAAATTGGTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATC

[0487] TCATGTTCATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATT

[0488] GACCCGTATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTT

[0489] CTATTCGAGATCTCCTCGACACCGCCTCTGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAACATT

[0490] GTTCACCTCACCATACAGCACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTGATGAATCTGGCCA

[0491] CCTGGGTGGGAAGTAATTTGGAAGACCCAGCATCCAGGGAATTAGTAGTCAGCTATGTCAATGTT

[0492] AATATGGGCCTAAAAATCAGACAACTACTGTGGTTTCACATTTCCTGTCTTACTTTTGGAAGAGAA

[0493] ACTGTTCTTGAGTATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCTGCTTACAGACCACCA

[0494] AATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGACGAGGCAGGTCCCCTAG

[0495] AAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTC

[0496] GGGAATCTCAATGTTAGTATCCCTTGGACTCATAAGGTGGGAAACTTTACTGGGCTTTATTCTTCT

[0497] ACTGTACCTGTCTTTAATCCTGAGTGGCAAACTCCCTCCTTTCCTCACATTCATTTACAGGAGGAC

[0498] ATTATTAATAGATGTCAACAATATGTGGGCCCTCTTACAGTTAATGAAAAAAGGAGATTAAAATT

[0499] AATTATGCCTGCTAGGTTCTATCCTAACCTTACCAAATATTTGCCCTTGGACAAAGGCATTAAACC

[0500] GTATTATCCTGAACATGCAGTTAATCATTACTTCAAAACTAGGCATTATTTACATACTCTGTGGAA

[0501] GGCTGGCATTCTATATAAGAGAGAAACTACACGCAGCGCCTCATTTTGTGGGTCACCATATTCTTG

[0502] GGAACAAGAGCTACAGCATGGGAGGTTGGTCTTCCAAACCTCGACAAGGCATGGGGACGAATCT

[0503] TTCTGTTCCCAATCCTCTGGGATTCTTTCCCGATCACCAGTTGGACCCTGCGTTCGGAGCCAACTC

[0504] AAACAATCCAGATTGGGACTTCAACCCCAACAAGGATCACTGGCCAGAGGCAAATCAGGTAGGA

[0505] GCGGGAGCATTCGGGCCAGGGTTCACCCCACCACACGGCGGTCTTTTGGGGTGGAGCCCTCAGGC

[0506] TCAGGGCATATTGACAACAGTGCCAGCAGCACCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAA

[0507] GACAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGGAACTCC

[0508] ACAACATTCCACCAAGCTCTGCTAGATCCCAGAGTGAGGGGCCTATATTTTCCTGCTGGTGGCTCC

[0509] AGTTCCGGAACAGTAAACCCTGTTCCGACTACTGCCTCACCCATATCGTCAATCTTCTCGAGGACT

[0510] GGGGACCCTGCACCGAACATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACA

[0511] GGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTC

[0512] TCTCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTCGCAGTCCCCAACCTCCAATCA

[0513] CTCACCAACCTCTTGTCCTCCAATTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATA

[0514] TTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGC

[0515] CCGTTTGTCCTCTACTTCCAGGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACGATT

[0516] CCTGCTCAAGGAACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACT

[0517] TGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTC

[0518] TCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTT

[0519] CAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTC

[0520] TATTACCAATTTTCTTTTGTCTTTGGGTATACATTTGAACCCTAATAAAACCAAACGTTGGGGCTA

[0521] CTCCCTTAACTTCATGGGATATGTAATTGGAAGTTGGGGTACTTTACCGCAAGAACATATTGTACT

[0522] AAAAATCAAGCAATGTTTTCGAAAACTGCCTGTAAATAGACCTATTGATTGGAAAGTATGTCAGA

[0523] GAATTGTGGGTCTTTTGGGCTTTGCTGCCCCTTTTACACAATGTGGCTATCCTGCCTTAATGCCTTT

[0524] ATATGCATGTATACAATCTAAGCAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTGTGTAA

[0525] ACAATATCTGAACCTTTACCCCGTTGCCCGGCAACGGTCAGGTCTCTGCCAAGTGTTTGCTGACGC

[0526] AACCCCCACTGGATGGGGCTTGGCTATTGGCCATCGCCGCATGCGTGGAACCTTTGTGGCTCCTCT

[0527] GCCGATCCATACTGCGGAACTCCTAGCAGCTTGTTTTGCTCGCAGCCGGTCTGGAGCGAAACTTAT

[0528] CGGAACCGACAACTCTGTTGTCCTCTCTCGGAAATACACCTCCTTTCCATGGCTGCTAGGGTGTGC

[0529] TGCCAACTGGATCCTGCGCGGGACGTCCTTTGTCTACGTCCCGTCGGCGCTGAATCCCGCGGACG

[0530] ACCCGTCTCGGGGCCGTTTGGGACTCTACCGTCCCCTTCTTCATCTGCCGTTCCGGCCGACCACGG

[0531] GGCGCACCTCTCTTTACGCGGTCTCCCCGTCTGTGCCTTCTCTCTGCCGGACCGTGTGCACTTCGC

[0532] TTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCAGGTCTTGCCCCAAGGTCTTACAT

[0533] AAGAGGACTCTTGGACTCTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTTT

[0534] GTTTAAGGACTGGGAGGAGTTGGGGGAGGAGATTAGGTTAATGATCTTGTACTAGGAGGCTGTA

[0535] GGCATAAATTGGTCTGTTCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCATGTTC

[0536] ATGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTGACCCGTA

[0537] TAAAGAATTGGAGCTTCTGTGGAGTTACTCTCTTTTTTGCCTTCTGACTTCTTTCCTTCTTATTCGA

[0538] GATCTCCTCCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCCGACTGTGCCTTCTAGT

[0539] TGCCAGCCATCTGTTGTTTGCCCCTCCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT

[0540] GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGTGTGTCATTCTATTCTGGGG

[0541] GGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT

[0542] GCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGC

[0543] GCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTG

[0544] CCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCC

[0545] CCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCC

[0546] CAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCC

[0547] CTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACC

[0548] CTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGA

[0549] GCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAA

[0550] GTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGT

[0551] GTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCA

[0552] ACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCG

[0553] CCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTC

[0554] CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTAT

[0555] ATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGAT

[0556] TGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACA

[0557] ATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAG

[0558] ACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCAC

[0559] GACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTAT

[0560] TGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC

[0561] ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGC

[0562] GAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGG

[0563] ACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGA

[0564] CGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCC

[0565] GCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTG

[0566] GCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGT

[0567] ATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGA

[0568] CTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACC

[0569] GCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAG

[0570] CGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTAC

[0571] AAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGT

[0572] TTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCG

[0573] TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA

[0574] GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT

[0575] GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAAC

[0576] GCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCT

[0577] CGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAA

[0578] TCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAA

[0579] AAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG

[0580] CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT

[0581] CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG

[0582] AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAA

[0583] GCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCT

[0584] TGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA

[0585] GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA

[0586] AGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC

[0587] TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA

[0588] GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA

[0589] AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAAT

[0590] TAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG

[0591] CTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCC

[0592] GTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG

[0593] AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC

[0594] AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA

[0595] AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC

[0596] TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC

[0597] ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC

[0598] AGTGTTATCACTCATGGTTATGGCAGCAC。

Claims

1. An isolated nucleic acid molecule, the nucleic acid sequence of which is shown in SEQ ID NO: 3 or 4.

2. A plasmid comprising the nucleic acid molecule of claim 1; preferably, the starting plasmid of the plasmid is pcDNA3.1(+), and the plasmid comprises 1.3 times the nucleic acid molecule of claim 1.

3. The plasmid according to claim 2, wherein The plasmid is B2-pcDNA3.1 or C2-pcDNA3.1, wherein the amino acid sequence of B2-pcDNA3.1 is shown in SEQ ID NO:5, and the amino acid sequence of C2-pcDNA3.1 is shown in SEQ ID NO:

6.

4. A cell comprising the nucleic acid molecule of claim 1 or the plasmid of claim 2 or 3.

5. The cell according to claim 4, wherein The host cell of the cell is HepG2 cell. 6 . A kit comprising the nucleic acid molecule of claim 1 , or the plasmid of claim 2 or 3 , or the cell of claim 4 or 5 .

7. A model for screening HBV drugs, comprising the nucleic acid molecule of claim 1, or the plasmid of claim 2 or 3, or the cell of claim 4 or 5, or the kit of claim 6.

8. Use of the nucleic acid molecule according to claim 1, or the plasmid according to claim 2 or 3, or the cell according to claim 4 or 5, or the kit according to claim 6, or the model according to claim 7 in screening HBV drugs.

9. A method for screening HBV drugs, comprising using the nucleic acid molecule of claim 1, or the plasmid of claim 2 or 3, or the cell of claim 4 or 5, or the kit of claim 6, or the model of claim 7.

10. The method according to claim 9, comprising: Cultivate the cells according to claim 4 or 5, administer the HBV drug to be screened, and detect the cell expression.

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