Universal bacterial artificial chromosome recombinant virus transfer vector and construction method thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a universal bacterial artificial chromosome recombinant virus transfer vector and its construction method. Background Technology

[0002] Bacterial artificial chromosomes, a vector system developed in the 1990s, were first used in the sequencing of the human genome. They offer advantages such as large capacity, stable genetic characteristics, and ease of manipulation. The application of bacterial artificial chromosome technology in viral genetics allows for the manipulation of viral genomes using bacterial genetics methods, opening new avenues for basic research in viral genetics. In recent years, while research on gene expression regulation, gene interaction, and target gene localization and transformation using bacterial artificial chromosomes as a technological platform has rapidly progressed in the post-genomic era, transfection of mammalian cells with molecularly cloned viral plasmids isolated from *E. coli* can initiate viral infection and produce pure recombinant viruses. This plasmid-based viral infectious cloning has many advantages: it is very stable in *E. coli*, conforms to *E. coli* gene manipulation techniques, and induces toxigenic replication after transfection without homologous recombination and sequence repair. Furthermore, it enables large-scale amplification of the viral genome in *E. coli* without the need for the emergence of true viruses, making it more suitable for convenient and safe gene manipulation and research on viruses with unclear biological backgrounds. Molecularly cloned viruses can be easily modified for homologous recombination. Many herpesviruses, such as human cytomegalovirus, herpes simplex virus, pseudorabies virus, Ebola virus, Marek's virus, bovine herpesvirus, mouse herpesvirus, varicella-zoster virus, and equine herpesvirus, have been reported to be constructed into bacterial artificial chromosomes.

[0003] Recently constructed artificial chromosomes for herpesviruses typically feature a loxp site flanking the core sequence, oriented in the same direction. This loxp site facilitates cleavage of the core sequence within eukaryotic cells, ensuring that the resulting recombinant virus exhibits biological characteristics more closely similar to the wild-type virus. The Cre-loxp recombinase system, first discovered in E. coli bacteriophages, consists of two parts: the Cre recombinase and the loxp sites. In aqueous solution, the enzyme exists as a monomer. The optimal reaction temperature for the recombinase is 37°C–42°C. It recognizes a 34 bp loxp sequence. In vivo and in vitro systems, the Cre enzyme induces four types of reactions between loxp sites: inversion, translocation, cleavage, and integration. When two sites are on the same molecule with opposite orientations of the spacer regions, an inversion occurs; when two sites are on the same molecule with the same orientation of the spacer regions, cleavage occurs; and when two sites are located on separate linear molecules, translocation occurs. These recombination reactions are all reversible.

[0004] According to literature reports, the actual screening process for LacZ and EGFP is quite cumbersome, hindering virus purification. Viral replication utilizes the purine metabolic pathway, which is blocked by mycophenolic acid; without this pathway, the virus cannot replicate normally. GPT can induce recombinant viruses to express xanthine-guanine phosphoribosyltransferase (XGPRT, gpt). The transferase itself does not remove the metabolic pathway blockade; once exogenous purine is added, exogenous salvage synthesis of guanine is completed, allowing the recombinant virus to replicate normally through the salvage pathway. Wild-type viruses, unable to use the salvage pathway, essentially die after several rounds of pressure screening, achieving enrichment and death of the recombinant virus. However, pressure screening cannot directly screen recombinant viruses, and its practical operation remains very difficult. Combining drug screening and fluorescent protein screening can improve the purification efficiency of recombinant viruses. Several studies have combined the two methods, but none have been successful; there are currently no publicly reported successful cases.

[0005] Generally, eukaryotic mRNA translation requires a 5' cap to mediate ribosome binding. These viruses lack a cap site before their mRNA but possess a 600-1200 bp 5' untranslated region containing multiple non-initiation AUGs. This long untranslated region is called the Internal Ribosome Entry Site (IRES). IRES recruits ribosomes to translate the mRNA. Fusion of IRES with exogenous cDNA has shown that IRES can independently initiate translation. Utilizing this property, IRES is now widely used in the construction of binary expression vectors. However, it is not a promoter in the traditional sense, and its translation efficiency is low. This is why IRES-EGFP, when used as a tracer, exhibits weak fluorescence and requires verification; it cannot be expressed under all circumstances. IRES does not have a strict fusion expression requirement; adding 10-15 bases immediately upstream or downstream will not substantially alter the downstream CDS translation.

[0006] Therefore, the development of a fast and simple operating system for reverse genetics of bacterial artificial chromosomes is of great and far-reaching significance for manipulating viral genomes and studying viral biological characteristics.

[0007] It should be noted that the information disclosed in this background section is only for understanding the background technology of the present application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention

[0008] This disclosure provides at least one universal bacterial artificial chromosome recombinant virus transfer vector and its construction method.

[0009] In a first aspect, embodiments of this disclosure provide a universal bacterial artificial chromosome recombinant viral transfer vector, the transfer vector comprising a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as the basic backbone and pUC19-BAC as the functional sequence.

[0010] In one optional embodiment, the pUC19-loxp-gpt-IRES-EGFP in the transfer vector comprises sequentially linked sequences of loxp, CMV promoter, gpt, IRES, EGFP, and SV40 terminator.

[0011] In one optional embodiment, the amino acid sequence of the gpt is SEQ ID NO.1, and the nucleotide sequence is SEQ ID NO.2.

[0012] In an optional embodiment, the nucleotide sequence of the IRES is SEQ ID NO.3.

[0013] In one optional embodiment, the amino acid sequence of the EGFP is SEQ ID NO.4, and the nucleotide sequence is SEQ ID NO.5.

[0014] Secondly, this disclosure also provides a method for constructing a universal bacterial artificial chromosome recombinant viral transfer vector as described above. The construction method of pUC19-loxp-gpt-IRES-EGFP is as follows: S11, designing a gpt-IRES-EGFP expression cassette: primers are designed to amplify gpt, IRES, and EGFP using a seamless cloning method to construct a complete dual expression reading frame. Simultaneously, primers are designed to amplify the CMV promoter and SV40 poly terminator sequences, introducing the CMV promoter and SV40 poly terminator upstream and downstream of the reading frame, respectively; S12, a loxp sequence site is introduced into the primer at the 5' end of the above-mentioned CMV promoter sequence; S13, a SmaI / SalI double restriction site is introduced upstream of the loxp at the 5' end of the gpt-IRES-EGFP expression cassette after S12 treatment, and three key restriction sites, SphI / BamHI / NotI, are introduced sequentially downstream of the expression cassette.

[0015] In an optional embodiment, the construction method of pUC19-BAC is as follows: S21, pBeloBAC11 is digested with SphI and ligated with pUC19 linearized plasmid digested with SphI using T4 Ligase, and then transformed into TransT1 competent cells; S22, the competent cells transformed in S21 are revived and plated on agar plates containing ampicillin and chloramphenicol resistance; S23, PCR identification is performed using universal primers M13-F / R to screen colonies with a band in the 7500bp range in S22; S24, the colonies suspected to be correctly identified in S23 are cultured overnight in a medium containing ampicillin and chloramphenicol; S25, a small amount of plasmid is extracted from the bacterial culture in S24, and the extracted plasmid is digested and identified. The correctly identified bacterial culture and plasmid are preserved for later use.

[0016] In an optional implementation, the construction of the transfer vector for viral recombination using a universal transfer vector is as follows: S31, selection of homologous arms: select upstream and downstream homologous arms of the target gene according to the viral genome characteristics, with a size of 1000-2000bp; S32, introduce SmaI and SalI restriction sites at both ends of the upstream homologous arm sequence selected in S31, and introduce BamHI and NotI restriction sites at both ends of the downstream homologous arm sequence; S33, introduce the upstream homologous arm sequence from S32 into the corresponding restriction site upstream of the gpt-IRES-EGFP expression cassette, and introduce the downstream homologous arm sequence into the corresponding restriction site downstream of the expression cassette; S34, after completely introducing the sequences of the homologous arms on both sides of the virus, introduce the BAC functional sequence into the SphI / BamHI restriction sites of the above vector.

[0017] In one optional embodiment, the steps for processing the viral genome using a viral recombinant transfer vector include: S41, co-transfecting the transfer vector and the viral genome into host cells, or transfecting host cells already infected with the virus; S42, selecting the host cells from S41 by pressure selection and fluorescent labeling to obtain purified recombinant virus; S43, expanding the culture of the purified recombinant virus and extracting the circularized viral genome, i.e., BACV; S44, electroporating the circularized viral genomic DNA into DH10B, and obtaining the correct strain through resistance selection and PCR identification; S45, expanding the culture of the BACV strain and extracting the BACV plasmid for verification; S46, transfecting the BACV plasmid from S45 into the host cells of the virus, culturing until cytopathic effects appear, harvesting the virus, and performing gene identification.

[0018] Thirdly, this disclosure also provides an application of the universal bacterial artificial chromosome recombinant virus transfer vector as described above in fields such as bacterial artificial chromosome manipulation of DNA virus genomes and construction of MDV viruses.

[0019] The beneficial effects of this invention are that the universal bacterial artificial chromosome recombinant viral transfer vector and its construction method connect the target gene reading frame for drug screening with the EGFP fluorescent protein gene reading frame using an IRES dual-expression ligation sequence, enabling dual screening. The BAC construction process separately constructs the screening gene and functional sequence into a high-copy T vector, facilitating replication and the preparation of sufficient plasmid DNA. Furthermore, the appropriate restriction enzyme sites facilitate the introduction of various homologous arms, ensuring broad targeting and enabling simultaneous manipulation of multiple genomic sequences, thus improving research efficiency.

[0020] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 A diagram illustrating the construction strategy of the universal construction vector pUC19-loxp-GPT-IRES-EGFP provided in this embodiment of the disclosure; Figure 2 A flowchart illustrating the construction process of the recombinant transfer vector provided in this embodiment of the disclosure; Figure 3 This is a diagram showing the PCR identification results of the pUC19-BAC plasmid provided in the embodiments of this disclosure; Figure 4 A diagram showing the PCR amplification results of seamless cloning-related fragments provided in the embodiments of this disclosure; Figure 5 This is a diagram showing the PCR amplification results of the left and right homologous arms of MDV US2 provided in this embodiment of the disclosure; Figure 6 A diagram showing the results of recombinant virus rescue provided in an embodiment of this disclosure. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] As used herein, the phrases “in one embodiment,” “according to one embodiment,” “in some embodiments,” etc., generally refer to the fact that a particular feature, structure, or characteristic following the phrase can be included in at least one embodiment of this disclosure. Therefore, a particular feature, structure, or characteristic can be included in more than one embodiment of this disclosure, such that these phrases do not necessarily refer to the same embodiment. As used herein, the terms “example,” “exemplary,” etc., are used to “serve as an example, instance, or illustration.” Any implementation, aspect, or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or superior to other implementations, aspects, or designs. Rather, the use of the terms “example,” “exemplary,” etc., is intended to present concepts in a specific manner.

[0026] In this document, exemplary embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings. As used herein, expressions such as “at least one of…” modify the entire list of elements when following a list of elements, rather than individual elements in the list. For example, the expression “at least one of a, b, and c” should be understood to include only a, only b, only c, both a and b, both a and c, both b and c, or all of a, b, and c.

[0027] The terminology used herein is for the purpose of describing specific exemplary configurations only and is not intended to be limiting. As used herein, the singular articles “a,” “an,” and “the” may also be intended to include plural forms unless otherwise clearly stated herein. The terms “comprising,” “including,” and “having” are inclusive and thus specify the presence of features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein should not be construed as requiring them to be performed in the specific order discussed or shown, unless specifically identified as such. Additional or alternative steps may be employed.

[0028] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0029] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0030] In a first aspect, embodiments of this disclosure provide a universal bacterial artificial chromosome recombinant viral transfer vector, the transfer vector comprising a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as the basic backbone and pUC19-BAC as the functional sequence.

[0031] In some embodiments, specifically, the pUC19-loxp-gpt-IRES-EGFP in the transfer vector comprises the ordered linkage of loxp, CMV promoter, gpt, IRES, EGFP and SV40 terminator sequences.

[0032] In some embodiments, specifically, the amino acid sequence of the gpt is SEQ ID NO.1, and the nucleotide sequence is SEQ ID NO.2; the gpt can enable the recombinant virus to express xanthine-guanine phosphoribosyltransferase. After the addition of exogenous purine, the recombinant virus can replicate normally through the rescue pathway, while the wild-type virus cannot use the rescue pathway and is basically dead after pressure screening, thus achieving the enrichment and death of the recombinant virus.

[0033] In some embodiments, specifically, the nucleotide sequence of the IRES is SEQ ID NO.3; the IRES can recruit ribosomes to translate mRNA. Utilizing this property, RES is currently widely used in the construction of binary expression vectors, although its translation efficiency is weaker than that of expression using CMV as a promoter.

[0034] In some embodiments, specifically, the amino acid sequence of the EGFP is SEQ ID NO.4, and the nucleotide sequence is SEQ ID NO.5; the EGFP produces green fluorescent protein, which can be used for tracing recombinant viruses.

[0035] This disclosure also provides a method for constructing a universal bacterial artificial chromosome recombinant viral transfer vector as described above. The construction method of pUC19-loxp-gpt-IRES-EGFP is as follows: S11, designing a gpt-IRES-EGFP expression cassette: primers are designed according to the seamless cloning method to amplify gpt, IRES and EGFP to construct a complete dual expression reading frame. At the same time, primers are designed to amplify the CMV promoter and SV40 poly terminator sequences, and the CMV promoter and SV40 poly terminator are introduced upstream and downstream of the reading frame, respectively; S12, a loxp sequence site is introduced into the primer at the 5' end of the above CMV promoter sequence; S13, a SmaI / SalI double restriction site is introduced upstream of the loxp at the 5' end of the gpt-IRES-EGFP expression cassette after S12 treatment, and three key restriction sites of SphI / BamHI / NotI are introduced sequentially downstream of the expression cassette.

[0036] In some embodiments, the specific construction method of pUC19-BAC is as follows: S21, pBeloBAC11 is digested with SphI and ligated with pUC19 linearized plasmid digested with SphI using T4 Ligase, and then transformed into TransT1 competent cells; S22, the competent cells transformed in S21 are revived and plated on agar plates containing ampicillin and chloramphenicol resistance; S23, PCR identification is performed using universal primers M13-F / R to screen colonies with a band of 7500bp in S22; S24, the colonies suspected to be correctly identified in S23 are cultured overnight in a medium containing ampicillin and chloramphenicol; S25, a small amount of plasmid is extracted from the bacterial culture in S24, and the extracted plasmid is digested and identified. The correctly identified bacterial culture and plasmid are preserved for later use.

[0037] In some embodiments, the specific construction operation of the transfer vector for viral recombination using a universal transfer vector is as follows: S31, selection of homologous arms: select upstream and downstream homologous arms of the target gene according to the viral genome characteristics, with a size of 1000-2000bp; S32, introduce SmaI and SalI restriction sites at both ends of the upstream homologous arm sequence selected in S31, and introduce BamHI and NotI restriction sites at both ends of the downstream homologous arm sequence; S33, introduce the upstream homologous arm sequence from S32 into the corresponding restriction site upstream of the gpt-IRES-EGFP expression cassette, and introduce the downstream homologous arm sequence into the corresponding restriction site downstream of the expression cassette; S34, after completely introducing the sequences of the homologous arms on both sides of the virus, introduce the BAC functional sequence into the SphI / BamHI restriction site of the above vector.

[0038] In some embodiments, the specific steps of using a viral recombinant transfer vector to extract the viral genome include: S41, co-transfecting the transfer vector and the viral genome into host cells, or transfecting host cells already infected with the virus; S42, selecting the host cells from S41 by pressure selection and fluorescent labeling to obtain purified recombinant virus; S43, expanding the culture of the purified recombinant virus and extracting the viral genome circularized into BACV; S44, electroporating the circularized viral genomic DNA into DH10B, and obtaining the correct strain through resistance selection and PCR identification; S45, expanding the culture of the BACV strain and extracting the BACV plasmid for verification; S46, transfecting the BACV plasmid from S45 into the host cells of the virus, culturing until cytopathic effects appear, harvesting the virus, and performing gene identification.

[0039] This disclosure also provides an application of the universal bacterial artificial chromosome recombinant virus transfer vector as described above in fields such as bacterial artificial chromosome manipulation of DNA virus genomes and construction of MDV viruses.

[0040] Example 1: Construction of the universal vector pUC19-loxp-gpt-IRES-EGFP Please see Figure 1 ,like Figure 1 As shown, the specific steps are as follows: Gpt sequence acquisition: The gpt sequence information, with a size of 459bp, was obtained through the reference sequence Expression vector pCR-Amp-mCherry-XGPRT DNA; the DNA sequence was obtained through gene synthesis.

[0041] Primer design: Primers for amplifying the target fragments CMV promoter, gpt, IRES, EGFP, and SV40poly were designed according to the instructions of the one-step seamless cloning kit from Nanjing Novizan Biotechnology Co., Ltd. Loxp and SalI restriction sites were introduced at the 5' end of CMV, and SphI restriction sites were introduced at the 3' end of SV40poly. The primers are: CMV-P1 / P2; gpt-P1 / P2; IRES-P1 / P2; EGFP-P1 / P2; SV40-P1; SV40-P1 / P2.

[0042] Target band amplification: Using pCI-neo and the synthesized gpt sequence as templates, the target fragments of CMV promoter, gpt and SV40poly were amplified, and the gene fragments of IRES and EGFP were amplified at the same time.

[0043] PCR amplification of linearized vector pUC19-liner: Reverse amplification primers were designed on both sides of the multiple cloning site to amplify the pUC19-liner band, which was then purified and its concentration was measured for later use.

[0044] One-step cloning: The target slice was cloned into the pUC19-liner vector and transformed into TransT1 competent cells according to the one-step cloning kit, and screened using an resistant culture plate containing Amp.

[0045] Identification: The following day, colonies were picked for PCR identification. Correctly identified colonies were cultured overnight in liquid medium containing Amp resistance. Plasmid was extracted using the Axygen mini plasmid extraction kit, named pUC19-loxp-gpt-IRES-EGFP, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing was successful, confirming the recombinant plasmid pUC19-loxp-gpt-IRES-EGFP.

[0046] Example 2: Construction of a vector containing the functional sequence pUC19-BAC Please see Figure 2 ,like Figure 2 As shown, the specific steps are as follows: pBeloBAC11 and pUC19 were digested with SphI and ligated. The ligation products were transformed into TransT1 competent cells and screened using resistant culture plates containing Amp and chloramphenicol. After PCR identification, plasmids were extracted and digested for identification. The correctly identified plasmids and bacterial cultures were preserved for later use.

[0047] Please see Figure 3 ,like Figure 3 As shown in the figure, M: DL15000 DNA Marker; 1: pUC19-BAC; 2: Empty vector control; 3: Negative control.

[0048] Example 3: Construction of the pUC19-loxp-gpt-IRES-EGFP-LR-BAC recombinant transfer vector Taking the Malik CVI988 virus genome as an example, the homologous arms flanking the target gene US2, which is not essential for viral replication, were selected, with a size of about 2000bp.

[0049] Primers were designed to amplify the left homologous arm L and the right homologous arm R, respectively. A SmaI restriction site was introduced at the 5' end and a SalI restriction site was introduced at the 3' end of the left homologous arm; a BamHI restriction site was introduced at the 5' end and a NotI restriction site was introduced at the 3' end of the right homologous arm.

[0050] Target fragment amplification: Homologous arm DNA fragments were obtained by PCR amplification. The target fragments were cloned into pEASY-blunt blunt ends and ligated. After transformation, the fragments were plated on AIX selection plates for blue-white screening.

[0051] Identification: Plasmids with white spots were picked and identified by PCR. The plasmids with correct sequences were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The plasmids with correct sequences were named pEASY-blunt-L and pEASY-blunt-R, respectively.

[0052] Construction of viral recombinant transfer vector: The plasmid containing the L homologous arm sequence was ligated with pUC19-loxp-gpt-IRES-EGFP after double digestion with SmaI and SalI. After transformation and identification, pUC19-loxp-gpt-IRES-EGFP-L was obtained. Then, the plasmid containing the R homologous arm was ligated with pUC19-loxp-gpt-IRES-EGFP-L after double digestion with BamHI and NotI. After transformation and correct identification, pUC19-loxp-gpt-IRES-EGFP-LR was obtained.

[0053] The pUC19-BAC and the aforementioned pUC19-loxp-gpt-IRES-EGFP-LR plasmid were digested with SphI and BamHI. The target fragments, BAC (approximately 7500 bp) and pUC19-loxp-gpt-IRES-EGFP-LR (approximately 4000 bp), were recovered. The purified fragments were ligated and transformed into DH10B competent cells. After verification, the viral recombinant transfer vector pUC19-loxp-gpt-IRES-EGFP-LR-BAC containing the functional sequence was obtained.

[0054] Please see Figure 4 ,like Figure 4 As shown, M: DL5000 DNA Marker; 1: GPT amplification fragment; 2: IRES amplification fragment; 3: Loxp-CMV amplification fragment; 4: SV40 Poly(A) Signal amplification fragment; 5: EGFP amplification fragment.

[0055] Example 4: Functional validation of the pUC19-loxp-gpt-IRES-EGFP-LR-BAC transfer vector Virus expansion culture: After the virus is taken out of the liquid nitrogen tank and revived, it is inoculated into CEF cells and cultured for 3-5 days until obvious lesions appear.

[0056] Viral genome extraction: Total viral DNA was extracted from the harvested viral cultures using DNA extraction phenol reagent.

[0057] Co-transfection: pUC19-loxp-gpt-IRES-EGFP-LR-BAC was co-transfected with viral genomic DNA into CEF cells and cultured for 5-7 days.

[0058] Virus purification and identification: On day 3 post-transfection, pressure screening was performed (mycophenolic acid: 25ug / ml; xanthine: 25ug / ml; hypoxanthine: 100ug / ml). After 6 rounds of pressure screening and picking of green fluorescent plaques, pure recombinant virus was obtained and identified by PCR as a recombinant virus containing gpt-IRES-EGFP-BAC.

[0059] Recombinant viral circular genome extraction: Total viral DNA was extracted from cells on day 1 after inoculation.

[0060] Preparation of artificial chromosomes from viruses and bacteria: Circular viral DNA was electroporated into DH10B competent cells, and recombinant bacteria rBAC-MDV (DH10B) was obtained after the genes were identified as correct.

[0061] Functional verification of recombinant viral plasmid: Recombinant bacteria rBAC-MDV (DH10B) were cultured in liquid and the rBAC-MDV plasmid was extracted by precipitation method. The rBAC-MDV plasmid was transfected into CEF cells. After culturing for 5-7 days, when obvious green fluorescent disease appeared, the culture was harvested and DNA was extracted for recombinant gene identification. It was identified as a recombinant virus.

[0062] Please see Figure 5 ,like Figure 5 As shown, M: DL5000 DNA Marker; 1: US2 left homologous arm; 2: US2 right homologous arm.

[0063] Please see Figure 6 ,like Figure 6 As shown, A: lesion; B: fluorescent plaque; C: normal cell.

[0064] The above experimental results show that the universal recombinant transfer vector constructed in this invention can rapidly prepare transfer vectors containing homologous arms of viral target genes, and successfully obtain bacterial chromosomes containing viral genomes through functional verification. Furthermore, the extracted rBAC-MDV can be used to transfect cells to obtain the desired chromosomes.

[0065] SEQ ID NO.1 153 PRT gpt amino acid sequence MSEKYIVTWDMLQIHARKLASRLMPSEQWKGIIAVSRGGLVPGALLARELGIRHVDTVCISSYDHDNQRELKVLKRAEGDGEGFIVIDDLVDTGGTAVAIREMYPKAHFVTIFAKPAGRPLVDDYVVDIPQDTWIEQPWDMGVVFVPPISGR. SEQ ID NO.2 459 DNA gpt nucleotide sequence ATGAGCGAAAAATACATCGTCACCTGGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGCCGACTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTGGCGGTCTGGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTCGTCATGTCGATACCGTTTGTATTTCCAGCTACGATCACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGATGGCGAAGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGCGGTTGCGATTCGTGAAATGTATCCAAAAGCGCACTTTGTCACCATCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGATGACTATGTTGTTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATGGGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTAA. SEQ ID NO.3 574 PRT IRES nucleotide sequence CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAA. SEQ ID NO.4 240 PRT Amino acid sequence of EGFP protein MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK. SEQ ID NO.5 1389 DNA Nucleotide sequence of EGFP protein . In summary, this universal bacterial artificial chromosome recombinant viral transfer vector and its construction method ligate the target gene reading frame for drug screening with the EGFP fluorescent protein gene reading frame using an IRES dual-expression ligation sequence, enabling dual screening. The BAC construction process separately constructs the screening gene and functional sequence into a high-copy T vector, facilitating replication and the preparation of sufficient plasmid DNA. Furthermore, the use of appropriate restriction enzyme sites facilitates the introduction of various homologous arms, ensuring broad targeting and enabling simultaneous manipulation of multiple genomic sequences, thus improving research efficiency.

[0066] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A universal bacterial artificial chromosome recombinant virus transfer vector, characterized in that, The transfer vector includes a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as the basic backbone and pUC19-BAC as the functional sequence.

2. The universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 1, characterized in that, The transfer vector pUC19-loxp-gpt-IRES-EGFP comprises the ordered ligation of loxp, CMV promoter, gpt, IRES, EGFP and SV40 terminator sequences.

3. The universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 1, characterized in that, The amino acid sequence of the gpt is SEQ ID NO.1, and the nucleotide sequence is SEQ ID NO.

2.

4. The universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 1, characterized in that, The nucleotide sequence of the IRES is SEQ ID NO.

3.

5. The universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 1, characterized in that, The amino acid sequence of the EGFP is SEQ ID NO.4, and the nucleotide sequence is SEQ ID NO.

5.

6. A method for constructing a universal bacterial artificial chromosome recombinant virus transfer vector as described in any one of claims 1-5, characterized in that, The construction method of pUC19-loxp-gpt-IRES-EGFP is as follows: S11, Design of gpt-IRES-EGFP expression cassette: Primers were designed to amplify gpt, IRES and EGFP using the seamless cloning method to construct a complete dual expression reading frame. At the same time, primers were designed to amplify the CMV promoter and SV40 poly terminator sequences, and the CMV promoter and SV40 poly terminator were introduced upstream and downstream of the reading frame, respectively. S12, introduce the loxp sequence site into the primer at the 5' end of the above CMV promoter sequence; S13 introduces a SmaI / SalI double restriction site upstream of the loxp at the 5' end of the gpt-IRES-EGFP expression cassette after S12 treatment, and introduces three key restriction sites, SphI / BamHI / NotI, downstream of the expression cassette in sequence.

7. The method for constructing a universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 6, characterized in that, The construction method of pUC19-BAC is as follows: S21, pBeloBAC11 was digested with SphI and then ligated with pUC19 linearized plasmid digested with SphI using T4 Ligase and transformed into TransT1 competent cells. S22: After the competent cells transformed in S21 were revived, they were plated on agar plates containing Amp and chloramphenicol resistance. S23 was identified by PCR using universal primers M13-F / R, and colonies with a band of 7500bp in S22 were screened out. S24, the colonies in S23 that were suspected to be correctly identified were cultured overnight in a medium containing ampicillin and chloramphenicol; S25 involves extracting a small amount of plasmid from the bacterial culture in S24, followed by enzyme digestion for identification. The correctly identified bacterial culture and plasmid are then preserved for future use.

8. The method for constructing a universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 6, characterized in that, The construction procedure for the transfer vector used for viral recombination using a universal transfer vector is as follows: S31, Selection of homologous arms: Select upstream and downstream homologous arms of the target gene based on viral genome characteristics, with a size of 1000-2000bp; S32 introduces SmaI and SalI restriction sites at both ends of the upstream homologous arm sequence selected in S31, and introduces BamHI and NotI restriction sites at both ends of the downstream homologous arm sequence. S33, except that the upstream homologous arm sequence of S32 is introduced into the corresponding restriction site upstream of the gpt-IRES-EGFP expression cassette, and the downstream homologous arm sequence is introduced into the corresponding restriction site downstream of the expression cassette. S34, after fully introducing the sequences of the homologous arms on both sides of the virus, introduces the BAC functional sequence into the SphI / BamHI restriction site of the above vector.

9. The method for constructing a universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 6, characterized in that, The steps involved in using a viral recombinant transfer vector to manipulate the viral genome include: S41, co-transfect host cells with the genome of the transfer vector and the virus, or transfect host cells that are already infected with the virus; S42, after pressure screening and fluorescent labeling screening of host cells in S41, yielded purified recombinant virus; S43, after the purified recombinant virus is expanded and cultured, the circularized genome of the virus, i.e., BACV, is extracted; S44, the circularized viral genomic DNA was electroporated into DH10B, and the correct strain was obtained through resistance screening and PCR identification; S45, expand the culture of BACV strain, and extract BACV plasmid for verification; S46: The BACV plasmid in S45 is transfected into the host cells of the virus, cultured until cytopathic effects appear, and the virus is harvested and its genes are identified.

10. The application of the universal bacterial artificial chromosome recombinant virus transfer vector as described in claim 1 in the fields of bacterial artificial chromosome manipulation of DNA virus genomes and construction of MDV virus.

Images