HIV-1 genotype drug resistance detection quality control product based on lentiviral vector system as well as preparation method and application of HIV-1 genotype drug resistance detection quality control product

Abstract

The invention relates to the technical field of lentiviral vectors. The invention provides an HIV-1 (human immunodeficiency virus-1) genotype drug resistance detection quality control product based on a lentiviral vector system as well as a preparation method and application of the HIV-1 genotype drug resistance detection quality control product. According to the invention, HIV-1 pseudovirus is packaged by using a four-plasmid lentiviral vector system, and a novel HIV-1 genotype drug resistance detection quality control product containing drug resistance mutation K103N is further prepared. The HIV-1 genotype drug resistance detection quality control product provided by the invention highly simulates the structure and characteristics of natural viruses, and is high in biological safety, sustainable to obtain and easy to prepare on a large scale. In addition, the HIV-1 genotype drug resistance detection quality control product provided by the invention also shows good uniformity and stability, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of lentiviral vectors, and in particular, to an HIV-1 genotype drug resistance detection quality control product based on a lentiviral vector system, a preparation method thereof, and an application thereof. Background Art

[0002] Combined antiretroviral therapy (cART) is currently the most effective method for treating AIDS. However, with the prolongation of the treatment time and the highly variable and rapidly replicating characteristics of HIV-1 itself, the problem of drug resistance has gradually become a challenge that must be faced during cART. To achieve efficient cART, accurate HIV-1 drug resistance detection is crucial. HIV-1 genotype drug resistance detection can timely evaluate the drug resistance status of patients with antiviral treatment failure, providing an important scientific basis for clinicians to adjust the medication plan. Currently, laboratories engaged in HIV-1 genotype drug resistance detection mainly use laboratory self-built methods for detection. Therefore, the detection ability and detection quality urgently need to be confirmed and supervised, and quality control carried out through HIV-1 genotype drug resistance detection quality control products is crucial.

[0003] However, currently, clinical samples of HIV-1 infected patients containing drug resistance mutations and drug-resistant strains are mostly used to prepare drug resistance quality control products. These methods have the following limitations: the sources of clinical samples are limited, the operations of virus strain isolation and culture are complex and have a high technical difficulty, and both have potential biosafety risks. Therefore, there is an urgent need to develop a sustainable and safe HIV-1 genotype drug resistance detection quality control product, which can be used for internal quality control and inter-laboratory quality assessment in HIV-1 drug resistance detection laboratories, discover and solve problems existing in the detection, and ensure the accuracy and reliability of the detection results.

[0004] Lentiviral vectors (LV) are recombinant retroviral vectors based on HIV-1 and have been widely used as viral vector tools. Pseudoviruses prepared based on the LV system can highly mimic the characteristics of natural viruses, have high biosafety, and can be operated in a biosafety level 2 laboratory, meeting the needs of most laboratories. Compared with clinical samples and drug-resistant strains, the LV system has unique gene editing advantages and can provide more diverse HIV-1 genotype drug resistance detection quality control products by designing and introducing multiple drug resistance mutations on the expression vector plasmid. In addition, the process of packaging pseudoviruses with the LV system is simple and easy to operate. Based on the above advantages, the present invention uses the LV system to establish and develop a new type of HIV-1 genotype drug resistance detection quality control product and a preparation method thereof. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing a quality control product for HIV-1 genotypic drug resistance detection based on a lentiviral vector system. The present invention uses an improved four-plasmid vector technology platform to successfully prepare a new quality control product for HIV-1 pseudovirus genotypic drug resistance detection containing the drug resistance mutation K103N. The quality control product for HIV-1 genotypic drug resistance detection provided by the present invention has good homogeneity and stability; can simulate the characteristics of natural viruses and achieve quality control of the entire nucleic acid detection process; can be prepared in large quantities at low cost; and quality control products containing specific drug resistance mutation sites can be prepared through gene editing according to the actual quality control requirements of detection. The present invention provides a reliable technical platform for preparing quality control products for HIV-1 pseudovirus genotypic drug resistance detection using a lentiviral vector system.

[0006] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant vector expression plasmid pLL3.7-U6-pol, characterized in that the nucleotide sequence of the recombinant vector expression plasmid is as shown in SEQ ID NO.1;

[0008] SEQ ID NO.1:

[0009]

[0010] The present invention also provides the use of the recombinant vector expression plasmid in the preparation of a lentiviral vector system.

[0011] The present invention also provides a method for constructing the recombinant vector expression plasmid, comprising the following steps:

[0012] (1) Using the pol region gene sequence 2051 - 4700nt containing the drug-resistant mutation K103N as the target sequence;

[0013] (2) Inserting the target sequence into the vector expression plasmid pLL3.7-U6-del to obtain the recombinant vector expression plasmid pLL3.7-U6-pol;

[0014] The nucleotide sequence of the 2051 - 4700nt of the pol region described in step (1) is as shown in SEQ ID NO.2;

[0015] SEQ ID NO.2:

[0016]

[0017] In step (2), the upstream primer and the downstream primer in the 2051-4700 nt of the pol region are used during insertion;

[0018] The nucleotide sequence of the upstream primer in the 2051-4700 nt of the pol region is shown in SEQ ID NO.3;

[0019] The nucleotide sequence of the downstream primer in the 2051-4700 nt of the pol region is shown in SEQ ID NO.4;

[0020] The nucleotide sequence of the vector expression plasmid pLL3.7-U6-del is shown in SEQ ID NO.5;

[0021] SEQ ID NO.5:

[0022]

[0023] The present invention also provides a lentiviral vector system, comprising the recombinant vector expression plasmid pLL3.7-U6-pol, backbone plasmids pRSV-Rev and pMDLgag-poL RRE, and envelope plasmid pMD2.G.

[0024] The present invention also provides a method for preparing HIV-1 pseudovirus using the lentiviral vector system, comprising the following steps:

[0025] (1) Transfect HEK293T cells using the HIV-1 lentiviral vector system. After mixing the plasmids with the transfection reagent and allowing them to stand, slowly add them to the culture medium.

[0026] (2) After transfection, culture for 44 - 52 h, and collect the culture supernatant.

[0027] (3) Centrifuge the culture supernatant and then filter it to obtain the HIV-1 pseudovirus supernatant.

[0028] In step (1), the transfection system used is: 7 - 9 μg of recombinant vector expression plasmid, 3 - 5 μg of pRSV-Rev, 3 - 5 μg of pMDLgag-pol RRE, and 3 - 5 μg of pMD2.G. Add 46 - 50 μL of Lipofectamine 2000 to 240 - 260 μL of Optimem (Gibco) and mix well, then transfect 0.8 - 1.2×10 7 cells / T75 of 293T cells;

[0029] In step (2), the culture time is 44 - 52 h;

[0030] In step (3), the centrifugation speed is 1800 - 2200 rpm, the centrifugation time is 8 - 12 min, and a 0.45 μm microporous filter is used for filtration.

[0031] The present invention also provides the HIV-1 pseudovirus prepared by the above method.

[0032] The present invention also provides the application of the HIV-1 pseudovirus in the preparation of HIV-1 genotype drug resistance detection quality control products.

[0033] The beneficial effects of the above technical solutions of the present invention are as follows:

[0034] (1) High biosafety

[0035] The present invention uses a four-plasmid lentiviral vector system to effectively prevent the "reappearance of infectivity" of pseudoviruses caused by carrying HIV-1 RNA fragments, and has high biosafety. Example data shows that the HIV-1 pseudoviruses used in the preparation of the HIV-1 genotype drug resistance detection quality control product of the present invention only have the ability of single-round infection. Compared with clinical samples and drug-resistant strains, the high biosafety of pseudoviruses effectively overcomes the problem of biosafety risks existing in clinical samples and drug-resistant strains, can be operated in a biosafety level 2 laboratory, reduces the biosafety level requirements for the operating environment and subsequent transportation and storage conditions, can meet the experimental needs of most laboratories, and is convenient for operation, transportation and storage.

[0036] (2) Highly simulate the biological characteristics of natural viruses

[0037] The pseudoviruses packaged by the lentiviral vector system can highly simulate the characteristics of natural viruses, so that like clinical samples and drug-resistant strains, they can simulate the detection process of clinical samples, and can effectively carry out the whole-process quality control of drug resistance detection to ensure the accuracy and reliability of the detection results.

[0038] (3) The preparation process is simple and it is easy to continuously obtain a large amount

[0039] Based on the lentiviral vector system provided by the present invention, the process of packaging pseudoviruses is simple, and the vector expression plasmid pLL3.7-U6-pol contains the cPPT / CTS sequence of the HIV-1 pol gene for the nuclear transport of the helper virus DNA, with high transfection efficiency and high titer of the packaging product. Example data shows that the HIV-1 pseudovirus packaging method of the present invention can obtain an HIV-1 pseudovirus supernatant with a titer of up to 10 9 copies / mL. Compared with clinical samples and drug-resistant strains, pseudoviruses are easier to prepare and continuously obtain a large amount, and are easy to store and transport.

[0040] (4) Have good quality control product performance

[0041] Example data shows that the quality control products prepared by the present invention have good uniformity; when stored at room temperature (25 °C), 4 °C and -20 °C for 28 days, the target sequence containing drug-resistant mutations can still be amplified, and it has good stability. Therefore, the HIV-1 genotype drug resistance detection quality control product provided by the present invention has good application value.

[0042] (5) Have unique gene editing advantages

[0043] Different from clinical samples and drug-resistant strains that can only carry specific drug-resistant mutations, the method for preparing a drug-resistant detection quality control product by the lentiviral vector system in the present invention has unique gene editing advantages. The present invention has successfully inserted a 2,648-bp foreign gene. Existing studies have shown that the lentiviral vector system can carry a foreign gene of about 8 kb. Based on the method provided by the present invention, multiple common drug-resistant mutations can be encapsulated into pseudovirus particles in the future, so as to prepare an HIV-1 pseudovirus genotype drug-resistant detection quality control product that meets the actual needs, providing a variety of quality control product options for drug-resistant detection quality control work. Description of the Drawings

[0044] Figure 1 Schematic diagram showing the positions of the pol region gene sequence of the insertion vector expression plasmid pLL3.7-U6-del and the HIV-1 genotype drug-resistant detection sequence in the HXB2 genome (the red straight line shows the target sequence, and the green straight line shows the HIV-1 genotype drug-resistant detection sequence).

[0045] Figure 2 Results diagram of the construction and identification of the recombinant vector expression plasmid pLL3.7-U6-pol; among them, A is the plasmid construction map of pLL3.7-U6-pol (red represents the target sequence, and dark green represents the GFP reporter gene); among them, B is the electrophoresis identification map after double digestion of pLL3.7-U6-pol plasmid and pLL3.7-U6-pol.

[0046] Figure 3 Representative fluorescence microscope images of the packaging and infection of HIV-1 pseudovirus; among them, A is the representative fluorescence microscope image after transfection of the recombinant vector expression plasmid pLL3.7-U6-pol into HEK-293T cells for 48 h; among them, B is the representative fluorescence microscope image after culturing the first-generation and second-generation culture supernatants with cells for 48 h.

[0047] Figure 4 Electrophoresis identification diagram of the target sequence inserted into the HIV-1 pseudovirus particles.

[0048] Figure 5 Electrophoresis result diagram for the evaluation of the homogeneity of the quality control product.

[0049] Figure 6 Viral load result diagram for the evaluation of the stability of the quality control product; among them, A, B, and C are the short-term stability viral load detection result diagrams under the conditions of room temperature (25 °C), 4 °C, and -20 °C respectively, and D is the freeze-thaw stability viral load detection result diagram after repeated freeze-thawing 1-5 times.

[0050] Figure 7It is an electrophoresis result diagram for the stability evaluation of quality control products; among them, A, B, and C are the short-term stability electrophoresis results under room temperature (25°C), 4°C, and -20°C conditions respectively, and D among them is the freeze-thaw stability electrophoresis result after repeated freeze-thaw cycles 1-5 times Detailed implementation manners

[0051] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention

[0052] Example 1 Material preparation

[0053] 1. Cell lines, plasmids and samples

[0054] HEK-293T cells (SCSP-502) were purchased from the Cell Bank of the Chinese Academy of Sciences

[0055] The 4 plasmids of the LV system include: backbone plasmids (pRSV-Rev and pMDLgag-pol RRE) and envelope plasmid (pMD2.G), and the Gibson seamless cloning kit was purchased from Beijing Bomed Company; the vector expression plasmid pLL3.7-U6-del was constructed and stored in this laboratory, and this plasmid contains green fluorescent protein (GFP) as a reporter gene

[0056] The samples used in the present invention are all from the sample library of the Reference Laboratory of the National Center for AIDS / STD Control and Prevention, Chinese Center for Disease Control and Prevention

[0057] 2. Main reagents

[0058] Lipofectamine 2000 transfection reagent was purchased from Thermo Company, restriction endonucleases were purchased from New England Biolabes (NEB) Company, PBS, FBS, DMEM and Opti-MEM were purchased from Gibco Company, QIAamp Viral RNA Mini Kit nucleic acid extraction kit and dPCR OneStep Advanced Probe digital PCR kit was purchased from Qiagen Company, One Step RNA PCR kit (AMV) was purchased from TaKaRa Company, 2×Taq plus PCR Mix kit was purchased from Beijing Tiangen Company, DAPI, Triton and 4% paraformaldehyde cell fixative were all purchased from Solarbio

[0059] Example 2 Selection of target sequence and construction of recombinant vector expression plasmid

[0060] 1. Selection and synthesis of target sequence

[0061] The inventors selected a 2648 bp pol region sequence (SEQ ID NO.2) from the HIV-1 CRF07_BC gene sequence of 1 case of treatment-failure-infected individuals as the target sequence of the present invention. This sequence contains the full length of protease (PR) and reverse transcriptase (RT) regions and a partial sequence of integrase (IN) region, and contains the drug-resistant mutation K103N. Taking HXB2 as a reference, the position of the target sequence is as Figure 1 shown. The above 2648 bp target sequence was entrusted to Bomed Company for gene synthesis and cloned into the cloning vector plasmid pUC57 to obtain the plasmid pUC57-pol containing the target sequence.

[0062] 2. Construction and identification of the recombinant vector expression plasmid pLL3.7-U6-pol

[0063] (1) Linearize the vector expression plasmid pLL3.7-U6-del

[0064] Use the restriction endonuclease XbaI to digest the vector expression plasmid pLL3.7-U6-del at 37 °C for 1 h. After the digestion is completed, heat-treat at 65 °C for 20 min, and run gel electrophoresis to recover and purify to obtain the linearized vector product pLL3.7-U6-del. The double digestion reaction system is shown in Table 1.

[0065] Table 1 Double digestion reaction system

[0066]

[0067] (2) Obtain a double-stranded DNA fragment containing the target sequence

[0068] Using the plasmid pUC57-pol as a template, use an upstream primer with an XbaI upstream homologous arm sequence (excluding the XbaI sequence) and a restriction endonuclease BamHI sequence, and a downstream primer with an XhoI downstream homologous arm sequence (including the XhoI sequence) to perform PCR amplification on the target sequence to obtain a double-stranded DNA fragment containing the target sequence.

[0069] The primer sequences are as follows, with the underlines indicating the sequences of the restriction endonucleases BamHI and XhoI:

[0070] F1: 5'-acagcagagatccagtttgg ggatcc accaaatgaaagattgt-3' (SEQ ID NO.3),

[0071] R1: 5'-ttaccgtaagttatgtaacg ctcgag ttctttattc-3' (SEQ ID NO.4).

[0072] The specific construction steps of the vector expression plasmid pLL3.7-U6-pol are as follows:

[0073] (3) Obtain the ligation product pLL3.7-U6-pol

[0074] The double-stranded DNA fragment obtained in step (2) was subjected to Gibson seamless cloning with the linearized vector pLL3.7-U6-del. The ligation reaction conditions were 50 °C for 30 min to obtain the ligation product pLL3.7-U6-pol.

[0075] The Gibson seamless cloning system is shown in Table 2.

[0076] Table 2 Gibson seamless cloning system

[0077]

[0078] (4) Transform the ligation product pLL3.7-U6-pol

[0079] The ligation product in step (3) was transformed into stble 3 competent cells. The specific steps are as follows:

[0080] Take 10 μL of the ligation product and add it to 100 μL of stble 3 competent cells. Incubate on ice for 30 min, heat shock in a water bath at 42 °C for 90 s, and then incubate on ice for 2 min. Add 500 μL of SOB culture medium for recovery (37 °C, shaker at 200 rpm for 1 h). Take 200 μL of the mixed culture medium and spread it on an Amp-resistant LB plate, and culture it overnight (15 h) in a 37 °C incubator.

[0081] (5) Obtain the recombinant vector expression plasmid pLL3.7-U6-pol

[0082] Pick monoclonal bacteria for small-scale plasmid extraction and send them for sequencing. Select the clones with correct sequencing for large-scale plasmid extraction to obtain the recombinant vector expression plasmid pLL3.7-U6-pol ( Figure 2 A).

[0083] The recombinant vector expression plasmid pLL3.7-U6-pol constructed above was verified by double digestion with BamHI and XhoI. The digestion system is shown in Table 3, digest at 37 °C for 1 h, and verify by gel electrophoresis.

[0084] Table 3 Double digestion reaction system

[0085]

[0086] The results are as Figure 2As shown in Figure B, the electrophoretic band position of the product obtained by double digestion of the recombinant vector expression plasmid pLL3.7-U6-pol was consistent with the expected result, and Sanger sequencing also proved the successful construction of the recombinant vector expression plasmid pLL3.7-U6-pol.

[0087] Example 3 Packaging and infection experiments of pseudoviruses

[0088] 1. Packaging and identification of pseudoviruses

[0089] (1) Packaging of HIV-1 pseudoviruses

[0090] Take 8 μL (1 μg / μL) of pLL3.7 or pLL3.7-U6-del, 4 μL (1 μg / μL) of pRSV-Rev, 4 μL (1 μg / μL) of pMDLgag-pol RRE, 4 μL (1 μg / μL) of pMD2.G and 50 μL of transfection reagent Lipofectamine 2000 and add them to 250 μL of Optimem (Gibco) and mix well. After standing at room temperature for 20 min, add the mixture to a T75 culture flask of HEK293T cells (1×10 7 cells / T75) containing 15 mL of 10% FBS DMEM medium and culture at 37 °C and 5% CO2. HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Catalog No.: SCSP-502).

[0091] (2) Harvesting of HIV-1 pseudovirus supernatant

[0092] After 4 h of transfection, replace with fresh 10% DMEM. After 48 h of transfection, collect the culture supernatant. Subsequently, perform DAPI nuclear staining. Observe the GFP fluorescence expression in cells using a fluorescence microscope, and untransfected cells were used as the negative control group.

[0093] After 48 h of transfection of the recombinant vector expression plasmid pLL3.7-U6-pol into HEK-293T cells, intracellular GFP fluorescence expression could be observed under a fluorescence microscope, and no fluorescence was seen in the negative control group ( Figure 3 A), indicating the successful packaging of HIV-1 pseudoviruses.

[0094] To remove cell debris, centrifuge the harvested culture supernatant at 2000 rpm at 4 °C for 10 min, and then filter it using a 0.45 μm microporous filter. Adjust the FBS concentration of the filtered supernatant to 20%, which is the pseudovirus supernatant, and aliquot it at 1 mL / tube and store it at -80 °C.

[0095] 2. Infection experiment of pseudoviruses

[0096] The HEK-293T cells were infected with the pseudovirus supernatant, and the uninfected cells were used as the negative control group. After 4 hours of infection, the cells were thoroughly washed with PBS solution and replaced with fresh 10% DMEM. After culturing for 48 hours, the GFP fluorescence expression was observed under a fluorescence microscope and the culture supernatant was harvested.

[0097] The culture supernatant was used to reinfect new HEK-293T cells. After culturing for 48 hours, the GFP fluorescence expression was observed under a fluorescence microscope.

[0098] After culturing the pseudovirus supernatant (the first generation) and the culture supernatant (the second generation) with cells for 48 hours respectively, GFP fluorescence expression was only observed in the HEK-293T cells infected with the pseudovirus supernatant, and no GFP fluorescence expression was detected in the HEK-293T cells infected with the culture supernatant. No fluorescence was seen in the two negative control groups of infection ( Figure 3 B). The above experimental results indicate that the HIV-1 pseudovirus can only be infected once, has no transmission risk, and has high biosafety.

[0099] Example 4 Identification of HIV-1 Pseudovirus

[0100] To verify whether the target sequence was successfully inserted into the HIV-1 pseudovirus particles, primer pairs F2 and R2 were designed at positions close to the target sequence on the recombinant vector expression plasmid pLL3.7-U6-pol. RNA was extracted from the HIV-1 pseudovirus supernatant, and the target sequence was amplified by RT-PCR and verified by gel electrophoresis and Sanger sequencing.

[0101] The upstream and downstream primer sequences are as follows:

[0102] F2: 5’-tgcaggggaaagaatagtagac-3’ (SEQ ID NO.6),

[0103] R2: 5’-agctctgcttatatagacct-3’ (SEQ ID NO.7).

[0104] Use The Viral RNA Mini Kit (purchased from QIAGEN / Qiagen) was used to extract 60 μL of RNA solution from 140 μL of the pseudovirus supernatant, and then a one-step RT-PCR kit (TaKaRa code: RR024A) was used for amplification detection. The detection program is shown in Table 4, and the reaction system is shown in Table 5.

[0105] Table 4 RT-PCR Reaction Conditions

[0106]

[0107]

[0108] Table 5 RT-PCR reaction system

[0109]

[0110] The gene sequence of the pol region inserted into the HIV-1 pseudovirus RNA was amplified by RT-PCR. The electrophoretic band position of the amplification product was around 3000 bp, which was consistent with the expected result ( Figure 4 ), and the Sanger sequencing result was also consistent with the target sequence, proving that the pol gene sequence was successfully inserted into the HIV-1 pseudovirus particles.

[0111] Example 5 Preparation of HIV-1 pseudovirus genotype drug resistance detection quality control product

[0112] In order to more accurately simulate clinical samples, the present invention uses HIV-1 negative plasma as a matrix to prepare an HIV-1 pseudovirus genotype drug resistance detection quality control product.

[0113] The inventors first used reverse transcription digital PCR (RT-dPCR) to quantitatively detect the HIV-1 pseudovirus supernatant. Using Viral RNA Mini Kit (purchased from QIAGEN / Kyowa), 50 μL of RNA solution was extracted from 140 μL of HIV-1 pseudovirus supernatant, and then OneStep Advanced Probe Kit was used for RT-dPCR detection, and the experimental operation was carried out strictly according to the instructions.

[0114] The process of the RT-dPCR detection is as follows:

[0115] Prepare the RT-dPCR amplification reaction system, and the reaction system is shown in Table 6.

[0116] Table 6 RT-dPCR amplification reaction system

[0117]

[0118]

[0119] The sequences of the upstream and downstream primers and probes in the reaction system:

[0120] F: 5’-gggagctctctggctaacta-3’ (SEQ ID NO.8),

[0121] R: 5’-ttaccagagtcacacaacagac-3’ (SEQ ID NO.9),

[0122] Probe: 5’FAM-tgccttgagtgcttcaagtagtgtgtg-3’BHQ1 (SEQ ID NO.10).

[0123] After the system is configured, place it in the QIAcuity one 5-plex real-time fluorescence quantitative PCR instrument (purchased from QIAGEN / Kaijie), select the FAM fluorescence signal acquisition channel, set conditions such as reaction temperature and cycle number, and the set parameters are shown in Table 7.

[0124] Table 7 RT-dPCR reaction conditions

[0125]

[0126] The quantification result of the HIV-1 pseudovirus supernatant is 1.25×10 9 copies / mL. The inventor diluted the HIV-1 pseudovirus supernatant to 10 4 copies / mL with HIV-1 negative plasma filtered through a 0.22μm filter, then oscillated overnight at 150 rpm at 4°C using a thermostatic oscillator, and aliquoted at 1 mL / tube and stored at -80°C for later use.

[0127] Example 6 Evaluation of the homogeneity and stability of the quality control product

[0128] According to the requirements of the GL003 guideline of the China National Accreditation Service for Conformity Assessment, randomly select 10 samples from the prepared quality control product, and repeat the detection of each sample 2 times for homogeneity evaluation, and use one-way ANOVA to evaluate the homogeneity; place the quality control product at room temperature (25°C), 4°C and -20°C for 0, 1, 3, 7, 14 and 28 days for short-term stability evaluation; place the quality control product stored at -80°C at room temperature (25°C) to melt and then freeze again at -80°C, and repeat the freeze-thaw 1 - 5 times for freeze-thaw stability evaluation. Detect 3 samples under different conditions of stability evaluation, use the RT-dPCR method to detect the viral load of the quality control product, and the results are expressed as log10 values, and use the HIV-1 drug resistance detection method to confirm the detection of drug resistance mutation sites.

[0129] 1. Results of homogeneity evaluation

[0130] Under the same conditions, randomly select 10 samples from the prepared quality control product to evaluate its homogeneity. Use RT-dPCR to detect the viral load of the quality control product, and the detection results are shown in Table 8. Calculate the average value of 20 detection results as 4.09 log10 copies / mL, and the F value is 0.049, which is less than the critical F threshold (F 0.05(9,10)= 3.02), and the difference was not statistically significant (P > 0.05). Further, drug resistance detection was performed on 10 quality control products, and the electrophoretic band positions of the amplified products of the drug resistance detection sequences were consistent with the expected results ( Figure 5 ), and the sequencing results also showed that drug resistance mutation K103N was detected in all 10 quality control products. The above results indicate that the quality control products have good homogeneity.

[0131] Table 8 Results of virus load detection for homogeneity evaluation of quality control products

[0132]

[0133] 2. Results of stability evaluation

[0134] The short-term stability and freeze-thaw stability studies were conducted to evaluate the stability of the quality control products under environmental conditions that may occur during transportation and storage. The RT-dPCR results showed that the overall trend of the virus load slightly decreased at room temperature (25 °C), and there was no obvious downward trend in the virus load under the conditions of 4 °C, -20 °C, or repeated freeze-thawing 5 times ( Figure 6 ). The drug resistance detection results showed that the electrophoretic band positions of the amplified products were consistent with the expected results when the quality control products were placed at room temperature (25 °C), 4 °C, -20 °C for 28 days or under the condition of repeated freeze-thawing 5 times ( Figure 7 ), and the drug resistance mutation K103N was stably detected by sequencing. The above results indicate that the quality control products have good stability.

[0135] As can be seen from the above embodiments, the present invention provides a method for preparing a quality control product for HIV-1 genotypic drug resistance detection based on a lentiviral vector system. The present invention uses an improved four-plasmid vector system to successfully prepare a novel HIV-1 pseudovirus genotypic drug resistance detection quality control product containing the drug resistance mutation K103N. The HIV-1 genotypic drug resistance detection quality control product prepared based on this improved system exhibits good homogeneity and stability. Using HIV-1 pseudovirus as a drug resistance detection quality control product in the present invention can not only highly simulate the structure and characteristics of natural viruses, but also improve the safety, sustainable availability, and preparation convenience of the drug resistance detection quality control product. The present invention provides a reliable technical platform for preparing HIV-1 pseudovirus genotypic drug resistance detection quality control products using a lentiviral vector system.

[0136] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant vector expression plasmid pLL3.7-U6-pol, characterized in that: The nucleotide sequence of the recombinant vector expression plasmid is shown in SEQ ID NO.

1.

2. Use of the recombinant vector expression plasmid according to claim 1 in preparing a lentiviral vector system.

3. The method for constructing the recombinant vector expression plasmid according to claim 1, characterized in that: The steps include: (1) The pol region gene sequence 2051-4700 nt containing the drug-resistant mutation K103N was used as the target sequence; (2) inserting the target sequence into the vector expression plasmid pLL3.7-U6-del to obtain the recombinant vector expression plasmid pLL3.7-U6-pol; The nucleotide sequence of 2051-4700 nt of the pol region in step (1) is shown in SEQ ID NO.2; The insertion in step (2) uses an upstream primer and a downstream primer of 2051-4700 nt of the pol region; The nucleotide sequence of the upstream primer of pol region 2051-4700 nt is shown in SEQ ID NO.3; The nucleotide sequence of the downstream primer of pol region 2051-4700 nt is shown in SEQ ID NO.4; The nucleotide sequence of the vector expression plasmid pLL3.7-U6-del is shown in SEQ ID NO.

5.

4. A lentiviral vector packaging system, characterized in that: It comprises the recombinant vector expression plasmid pLL3.7-U6-pol, backbone plasmids pRSV-Rev and pMDLgag-poLRRE, and envelope plasmid pMD2.G as described in claim 1.

5. A method for preparing HIV-1 pseudovirus using the lentiviral vector packaging system of claim 4, characterized in that: The steps include: (1) using the lentiviral vector packaging system of claim 4 to transfect HEK293T cells, mixing the plasmid and the transfection reagent, letting them stand, and then slowly adding them to the culture medium; (2) After transfection, culture for 44 to 52 h and collect the culture supernatant; (3) centrifuging and filtering the culture supernatant to obtain the HIV-1 pseudovirus supernatant; The transfection system used in step (1) is: 7-9 μg recombinant vector expression plasmid, 3-5 μg pRSV-Rev, 3-5 μg pMDLgag-pol RRE and 3-5 μg pMD2.G, 46-50 μL Lipofectamine 2000 is added to 240-260 μL Optimem (Gibco) and mixed, and 0.8-1.2×10 7 cells / T75 293T cells; The culturing time in step (2) is 44 to 52 hours; The centrifugal speed in step (3) is 1800-2200 rpm, the centrifugal time is 8-12 min, and a 0.45 μm microporous filter is used for filtration.

6. HIV-1 pseudovirus prepared by the method according to claim 5.

7. Use of the HIV-1 pseudovirus according to claim 6 in the preparation of quality control products for HIV-1 genotype drug resistance detection.

8. A quality control product for HIV-1 genotype drug resistance detection prepared using the HIV-1 pseudovirus according to claim 6.

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