WO2021212279A1 - 慢病毒的滴度提高型转移质粒 - Google Patents

慢病毒的滴度提高型转移质粒 Download PDF

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WO2021212279A1
WO2021212279A1 PCT/CN2020/085657 CN2020085657W WO2021212279A1 WO 2021212279 A1 WO2021212279 A1 WO 2021212279A1 CN 2020085657 W CN2020085657 W CN 2020085657W WO 2021212279 A1 WO2021212279 A1 WO 2021212279A1
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transfer plasmid
plasmid
virus
nucleic acid
hiv
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French (fr)
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杨小丹
陈世优
朱秀琴
陈超
陈小锋
李文佳
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广东东阳光药业有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to isolated nucleic acids, transfer plasmids, virus packaging kits, and methods for packaging viruses.
  • lentivirus is widely used in gene therapy for various diseases, such as CAR-T preparation and hematopoietic stem cell gene transduction.
  • the production process of lentivirus generally includes: 1 plasmid (including a transfer plasmid and helper plasmid) extraction and purification ⁇ 2 plasmid co-transfection into 293T or 293F cells ⁇ 3 collection of virus supernatant ⁇ 4 purification, concentration and sterility ⁇ 5 Get a lentivirus that can be used in gene therapy.
  • step 3 the initial titer of lentivirus production obtained in step 3 is low, and the available lentivirus yield obtained in step 5 is also extremely low (only about 20%); and step 2 uses instantaneous transfection.
  • the way of dyeing is high in production cost, and it is extremely difficult to expand the system. All these have resulted in a very limited production of lentivirus in a single batch, which cannot meet the treatment needs of indications with high viral vector dosages.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present invention proposes an isolated nucleic acid.
  • the nucleic acid has a nucleotide sequence shown in X 1 X 2 TAX 5 X 6 X 7 TX 9 , wherein X 1 is A or G; X 2 is T or C; X 5 is A or G; X 6 is C or T; X 7 is T or missing; X 9 is T or G; but does not have the nucleotide sequence shown in GTTAACTTT.
  • the isolated nucleic acid according to the embodiment of the present invention is located at the 5'end and 5'upstream of the cPPT/CTS element of the lentiviral packaging transfer plasmid. The transfer plasmid with the above isolated nucleic acid enters the cell nucleus efficiency greatly, and the packaged virus drops The degree is greatly improved.
  • the aforementioned nucleic acid may further include at least one of the following additional technical features:
  • the present invention has the nucleotide sequence shown in ACTAGTTG.
  • the titer of the virus packaged by the transfer plasmid of the nucleic acid with the nucleotide sequence shown by ACTAGTTG increased significantly.
  • the present invention proposes a transfer plasmid.
  • the transfer plasmid carries the aforementioned nucleic acid.
  • the titer of the virus packaged by the transfer plasmid according to the embodiment of the present invention is significantly improved. Compared with the prior art, the titer of the virus can be increased by at least 1.3 times.
  • the above-mentioned transfer plasmid may also include at least one of the following additional technical features:
  • it is located at the 5'end and upstream of the 5'end of the cPPT/CTS element in the transfer plasmid.
  • the nucleic acid is located within the range of 2 to 5 bp from the 5'end and 4 to 8 bp upstream of the 5'end of the cPPT/CTS element in the transfer plasmid.
  • the nucleic acid is located in the transfer plasmid Within the range of 3bp at the 5'end and 6bp upstream of the 5'end of the cPPT/CTS element.
  • the transfer plasmid has the nucleotide sequence shown in SEQ ID NO:1.
  • the virus packaged by the transfer plasmid with the nucleotide sequence shown in SEQ ID NO:1 can increase the virus titer by 1.3 to 1.5 times compared with the prior art.
  • the transfer plasmid includes an RSV promoter region and an HIV-1 5'LTR region, the promoter region is operably linked to the HIV-1 5'LTR region, and the HIV-1 5' The 5'end of the LTR region has a single ornithine acid.
  • the HIV-1 5'LTR region of the transfer plasmid is located at the 5'end of the HIV-1 genome, and a single guanine RNA is more likely to form a dimeric gRNA structure, which is encapsulated in viral particles to form a mature The RNA of 2 and 3 guanine is more likely to be used as mRNA to recruit ribosomes for translation.
  • the transfer plasmid according to the embodiment of the present invention produces more complete virus particles packaged with gRNA, and further improves the biological titer of the initial product of the lentivirus.
  • the transfer plasmid has the nucleotide sequence shown in SEQ ID NO: 2.
  • the virus packaged by the transfer plasmid having the nucleotide sequence shown in SEQ ID NO: 2 can increase the virus titer by 2-3 times compared with the prior art.
  • the present invention proposes a transfer plasmid.
  • the transfer plasmid has at least one of the following mutations: base 1569 is changed from G to A; base 1570 is changed from T The mutation is C; the 1573th base is mutated from A to G; the 1574th base is mutated from C to T; the 1575th base is deleted; the 1577th base is mutated from T to G.
  • the plasmid map of the pLV-mCherry is shown in FIG. 1, and the nucleotide sequence of the wild-type pLV-mCherry plasmid is shown in SEQ ID NO: 3.
  • SEQ ID NO: 3 is the first HIV-1 genome sequence (among which, GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGGATTTGAAATg truncated human terminal truncated HIV-1 Human immunodeficiency virus-1) region sequence, CTCTCTCGACGCA GGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGATGGGTGCGAGAGCGTC viral packaging signal ⁇ domain sequence,), GATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCA
  • the 5'end of the HIV-1 5'LTR region of the transfer plasmid lacks 2 GGs. This further increases the titer of the packaged virus.
  • the transfer plasmid has the nucleotide sequence described in SEQ ID NO: 1 or 2.
  • the present invention provides a virus packaging kit.
  • the kit includes the aforementioned transfer plasmid. Using the kit according to the embodiment of the present invention to package the virus, the titer of the virus is significantly improved.
  • the kit further includes a helper plasmid.
  • the present invention proposes a method of packaging viruses.
  • the method includes transfecting virus packaging tool cells with a virus packaging plasmid; incubating the transfected virus packaging tool cells to obtain a virus, wherein the virus packaging plasmid includes the aforementioned The transfer plasmid and helper plasmid mentioned above.
  • the titer of the packaged virus is significantly improved.
  • the virus packaging tool cell is 293T or 293F.
  • 293T or 293F is convenient for culture, rapid replication, and easy for transfection, which further increases the virus titer.
  • the “transfer plasmid” mentioned in this application refers to a plasmid that carries the target gene to be transduced and the sequence information of the HIV-1 regulatory cis-elements, such as pLV-mCherry, which is packaged in the virus.
  • the RNA produced by transcription in the cell can be packaged into a lentiviral vector;
  • the "helper plasmid” mentioned in this application refers to a plasmid carrying HIV-1 gag/pol, rev or envelope protein sequence information, except for the transfer plasmid.
  • this type of plasmid participates in the life cycle of the lentiviral vector in the form of protein after being transcribed and translated in the viral packaging cell.
  • Figure 1 is a plasmid map of pLV-mCherry according to an embodiment of the present invention
  • Figure 2 is a schematic diagram of pLV-mCherry transfer plasmid and its mutants according to an embodiment of the present invention
  • Figure 3 is a comparison diagram of lentivirus titers packaged by a three-plasmid system according to an embodiment of the present invention.
  • Fig. 4 is a comparison diagram of the titers of lentivirus packaged by a four-plasmid system according to an embodiment of the present invention.
  • sequence of the transfer plasmid shown in SEQ ID NO: 1 to 4 described in this application is the sequence displayed from the 5'end of the HIV-1 5'LTR region as the starting point, and is the same as SEQ ID NO: 1 to 1. 4 Transfer plasmids with identity are all within the protection scope of this application. At the same time, those skilled in the art understand that the plasmid has a circular structure, and the sequence of the plasmid can be displayed starting from the 5'end at any position.
  • sequence of the transfer plasmid displayed with the other 5'end as the starting point if it is different from SEQ ID NO:1 ⁇ 4, only the display starting point is different, but the sequence is the same, or the display starting point is different, but the sequence is the same as SEQ ID NO:1 ⁇ 4 sexuality is also within the scope of protection of this application.
  • the "identity” in this application refers to having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the sequence of this application.
  • the term percentage (%) "sequence identity" describes the number of matches ("hits") of identical nucleotides in two or more nucleic acid sequences in the alignment relative to the total length of the nucleic acid constituting the template. In other words, by using alignment, for more than two sequences or subsequences, these (sub)sequences are compared and aligned to obtain the results measured by a sequence comparison algorithm known in the art on the comparison window or designated area. When the maximum correspondence, or during manual comparison and visual inspection, the percentage of identical nucleotides can be determined
  • the purpose of the present invention is to provide three novel transfer plasmids for producing high-titer HIV-1 based lentiviral vectors.
  • the three plasmids are:
  • a new type of transfer plasmid for the production of high-titer HIV-1-based lentiviral vectors (named ⁇ GG or pLV- ⁇ GG(1-2)-mCherry plasmid in this application) (sequence is shown in SEQ ID NO: 4 Show)
  • deletion mutation is performed at the 5'end of the coding region of the lentiviral HIV-1 genome, and only one guanylic acid is retained, which increases the lentiviral biological titer by 1.5 to 2 times.
  • a new type of transfer plasmid for the production of high-titer HIV-1 based lentiviral vectors (named Mut or pLV-Mut(1569-1577)-mCherry plasmid in this application) (sequence is shown in SEQ ID NO:1) ,
  • the 9 bases (GTTAACTTT) near the 5'end and 5'end of the lentiviral cPPT/CTS element were mutated to 8 bases (ACTAGTTG), which increased the biological titer of the lentiviral vector to 1.3-1.5 times of the original .
  • a new type of transfer plasmid for producing high-titer HIV-1 based lentiviral vector (named ⁇ GG&Mut in this application or pLV- ⁇ GG(1-2)-Mut(1569-1577)-mCherry plasmid) (sequence (As shown in SEQ ID NO: 2), two mutations were introduced into the coding region of the lentiviral genome at the same time.
  • the mutation of 9 bases (GTTAACTTT) at and near the'end and 8 bases (ACTAGTTG) increases the biological titer of lentivirus to 2-3 times.
  • the novel transfer plasmid obtained by the present invention has inherent advantages in the ability to produce lentiviral vectors, and fundamentally improves the yield of the lentiviral vectors.
  • the mutation in the ⁇ GG mutant is to apply the replication mechanism of HIV-1 reported in the literature to the production of lentiviral vectors to increase the production of slow disease vectors.
  • the HIV-1-based lentiviral vector used retains the cis-acting elements of packaging, reverse transcription and integration in the HIV-1 genome, and these elements are used to regulate the transduction of foreign target genes. Therefore, the virus packaging, reverse transcription and integration methods are still consistent with HIV-1.
  • the genome of HIV-1 is positive-strand RNA.
  • the full-length positive-strand RNA can be used as messenger RNA (mRNA) in monomer form for translation, or it can form dimers as genomic RNA (gRNA) and be packaged into virus particles.
  • mRNA messenger RNA
  • gRNA genomic RNA
  • the virus regulates the replication cycle process of the virus by regulating the ratio of monomer and dimer. Siarhei et al.
  • the number of guanines at the 5'end of the full-length HIV-1 RNA is 1-3, and a single guanine RNA is more likely to form a dimeric gRNA structure, which is packaged into virus particles to form mature virus particles; and 2
  • One and three guanine RNAs are more likely to be used as mRNA to recruit ribosomes for translation.
  • the currently used HIV-1-based lentiviral vector transfer plasmids encode RNA starting positions with 3 guanines, which tend to be translated, but are not conducive to assembly into virus particles. Indeed, in the packaging process of lentivirus, a large number of empty shell viruses that are not packaged into gRNA will be produced.
  • lentiviral vector packaging is to produce more complete virus particles packaged with gRNA, indicating that more complete virus particles can be obtained by increasing the ratio of dimer gRNA, and the biological titer of the initial product of the lentivirus can be increased.
  • the Mut mutant is a transfer plasmid that the inventor of the present application unexpectedly discovered in scientific research that can significantly increase the titer of the packaged lentivirus.
  • ⁇ GG&Mut mutant is the introduction of the above two mutations into the lentiviral genome at the same time.
  • the two mutations can synergistically increase the titer of the lentivirus, resulting in a new type of transfer plasmid that produces high titer HIV-1 based lentiviral vectors.
  • a mutant transfer plasmid pLV- ⁇ GG(1-2)-mCherry plasmid containing GG(1-2) deletion mutation, where ⁇ GG(1-2) represents the transfer plasmid pLV-mCherry (abbreviated as WT, Fig. 2A)
  • WT transfer plasmid
  • Fig. 2B The first to second base pairs of the coding region of the HIV genome are deleted.
  • the above sequence was synthesized by a gene synthesis service company.
  • Respectively GG(1-2)-forward-primer-1 and GG(1-2)-rerward-primer-2, GG(1-2)-forward-primer-2 and GG(1-2)-rerward- primer-1 is a primer
  • pLV-mCherry is used as a template to obtain product fragments A and B by PCR. Recover fragments A and B products, then use the mixture of fragments A and B as templates, GG(1-2)-forward-primer-1 and GG(1-2)-rerward-primer-1 as primers, and pass over-lapping PCR Way to obtain product fragment C.
  • fragment C was ligated between the corresponding restriction sites of pLV-mCherry plasmid vector, and the vector was transformed into E. coli stbl3 competent. After restriction enzyme digestion and sequencing, the correct pLV- ⁇ GG(1-2) was obtained.
  • -mCherry plasmid (abbreviated as ⁇ GG, as shown in Figure 2B).
  • the above sequence was synthesized by a gene synthesis service company.
  • Respectively Mut(1569-1577)-forward-primer-1 and Mut(1569-1577)-rerward-primer-2 is a primer
  • Mut(1569-1577)-forward-primer-2 and Mut(1569-1577)-rerward- primer-1 is a primer
  • pLV-mCherry is used as a template to obtain product fragments D and E by PCR.
  • the products of fragment D and E are recovered, and then the mixture of fragment D and E is used as a template
  • Mut(1569-1577)-forward-primer-1 and Mut(1569-1577)-rerward-primer-1 are used as primers, and over-lapping PCR is performed Way to obtain product fragment F.
  • fragment F was ligated between the corresponding restriction sites of pLV-mCherry plasmid vector, and the vector was transformed into E. coli stbl3 competent.
  • the correct pLV-Mut (1569-1577)- mCherry plasmid (abbreviated as Mut, as shown in Figure 2C).
  • Example 1 and Example 2 The plasmids constructed in Example 1 and Example 2 were digested with SphI and NotI, respectively, and small fragments of ⁇ GG and large fragments of Mut were recovered. The two recovered fragments were ligated with T4 ligase and transformed into E. coli stbl3 competent. After restriction enzyme digestion and sequencing, the correct pLV- ⁇ GG(1-2)-Mut(1569-1577)-mCherry plasmid (referred to as ⁇ GG&Mut) was obtained. , As shown in Figure 2D).
  • Example 2 Take the plasmids constructed in Example 1 and Example 2, use the pLV-mCherry transfer plasmid as the control group, use psPAX2 and pMD2.G as the auxiliary plasmids, and use the three-plasmid system for lentivirus packaging.
  • the plasmid was transfected into 293T cells with lipofect3000, the virus supernatant from the cell culture was collected 50 hours after transfection, and filtered through a 0.45 ⁇ m filter. Perform titer determination.
  • the results are shown in Figure 3, the titers of lentivirus obtained in the Mut and ⁇ GG&Mut groups were significantly higher than those obtained by pLV-mCherry.
  • Example 2 Take the plasmids constructed in Example 1 and Example 2, use the pLV-mCherry transfer plasmid as the control group, and pMDLg-pRRE, pRSV-rev and pMD2.G as the auxiliary plasmids, and use the four-plasmid system for lentivirus packaging.
  • the plasmid was transfected into 293T cells with lipofect3000, and the cell culture supernatant was collected 50 hours after transfection. After the filter head is filtered, the titer is measured.
  • the results are shown in Figure 4, the titers of lentivirus obtained in the Mut and ⁇ GG&Mut groups were significantly higher than those obtained by pLV-mCherry.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.

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Abstract

一种分离的核酸。该核酸具有X 1X 2TAX 5X 6X 7TX 9所示的核苷酸序列，其中，X 1为A或G；X 2为T或C；X 5为A或G；X 6为C或T；X 7为T或缺失；X 9为T或G；但不具有GTTAACTTT所示的核苷酸序列。分离的核酸位于慢病毒包装转移质粒的cPPT/CTS元件的5'端及5'上游，具有上述分离的核酸的转移质粒进入细胞核效率大幅提高，所包装出的病毒的滴度大幅提高。

Description

慢病毒的滴度提高型转移质粒 技术领域
本发明涉及生物技术领域，具体地，本发明涉及分离的核酸、转移质粒、病毒包装试剂盒以及包装病毒的方法。
背景技术
目前，慢病毒作为一种基因递送载体，被广泛的应用于对各种疾病进行基因治疗，如CAR-T制备、造血干细胞基因转导。
慢病毒的生产工艺流程一般包括：①质粒(包括1个转移质粒和辅助质粒)提取和纯化→②质粒共转染293T或293F细胞→③收集病毒上清→④纯化、浓缩、无菌→⑤得到可供基因治疗使用的慢病毒。
现有的技术中，步骤③中得到的慢病毒生产初始滴度低、经步骤⑤得到的可供使用的慢病毒收率也极低(仅20％左右)；而步骤②中是采用瞬时转染的方式，生产成本高，且体系扩大难度极大。这些都导致单批次慢病毒产量十分有限，无法满足高病毒载体用量适应症的治疗需求。
因此，迫切需要开发出新的提高慢病毒产量的技术。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。
为此，在本发明的第一方面，本发明提出了一种分离的核酸。根据本发明的实施例，所述核酸具有X 1X 2TAX 5X 6X 7TX 9所示的核苷酸序列，其中，X 1为A或G；X 2为T或C；X 5为A或G；X 6为C或T；X 7为T或缺失；X 9为T或G；但不具有GTTAACTTT所示的核苷酸序列。根据本发明实施例的分离的核酸位于慢病毒包装转移质粒的cPPT/CTS元件的5’端及5’上游，具有上述分离的核酸的转移质粒进入细胞核效率大幅提高，所包装出的病毒的滴度大幅提高。
根据本发明的实施例，上述核酸还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，具有ACTAGTTG所示的核苷酸序列。具有ACTAGTTG所示的核苷酸序列的核酸的转移质粒所包装出的病毒滴度提高显著。
在本发明的第二方面，本发明提出了一种转移质粒。根据本发明的实施例，所述转移质粒携带前面所述的核酸。根据本发明实施例的转移质粒所包装出的病毒的滴度得到显著提高，相比于现有技术，病毒滴度至少可提高1.3倍。
根据本发明的实施例，上述转移质粒还可以包括如下附加技术特征至少之一：
根据本发明的实施例，位于所述转移质粒中cPPT/CTS元件的5’端及5’端上游。
根据本发明的实施例，所述核酸位于所述转移质粒中cPPT/CTS元件的5’端2～5bp及5’端上游4～8bp范围内，优选地，所述核酸位于所述转移质粒中cPPT/CTS元件的5’端3bp及5’端上游6bp范围内。
根据本发明的实施例，所述转移质粒具有SEQ ID NO:1所示的核苷酸序列。
Figure PCTCN2020085657-appb-000001
Figure PCTCN2020085657-appb-000002
Figure PCTCN2020085657-appb-000003
Figure PCTCN2020085657-appb-000004
根据本发明实施例的上述具有SEQ ID NO:1所示核苷酸序列的转移质粒所包装出的病 毒，相比于现有技术，病毒滴度可提高1.3～1.5倍。
根据本发明的实施例，所述转移质粒包含RSV启动子区和HIV-1 5’LTR区，所述启动子区与HIV-1 5’LTR区可操作地连接，所述HIV-1 5’LTR区的5’端具有单个鸟甘酸。根据本发明实施例的转移质粒的HIV-1 5’LTR区位于HIV-1基因组的5’端，而单个鸟嘌呤RNA更加倾向于形成二聚体gRNA结构，被包裹入病毒颗粒中，形成成熟的病毒颗粒，2个和3个鸟嘌呤的RNA则更倾向于作为mRNA招募核糖体等进行翻译。根据本发明实施例的转移质粒，相比于现有技术，会产生更多的包装有gRNA的完整病毒颗粒，进一步提高慢病毒初始产物的生物滴度。
根据本发明的实施例，所述转移质粒具有SEQ ID NO:2所示的核苷酸序列。
Figure PCTCN2020085657-appb-000005
Figure PCTCN2020085657-appb-000006
Figure PCTCN2020085657-appb-000007
Figure PCTCN2020085657-appb-000008
Figure PCTCN2020085657-appb-000009
根据本发明实施例的上述具有SEQ ID NO:2所示核苷酸序列的转移质粒所包装出的病毒，相比于现有技术，病毒滴度可提高2-3倍。
在发明的第三方面，本发明提出了一种转移质粒。根据本发明的实施例，相比于野生型pLV-mCherry(简称WT)，所述转移质粒具有下列突变的至少之一：第1569位碱基由G突变为A；第1570位碱基由T突变为C；第1573位碱基由A突变为G；第1574位碱基由C突变为T；第1575位碱基缺失；第1577位碱基由T突变为G。
根据本发明的实施例，所述pLV-mCherry的质粒图谱如图1所示，所述野生型pLV-mCherry质粒的核苷酸序列如SEQ ID NO:3所示。
Figure PCTCN2020085657-appb-000010
Figure PCTCN2020085657-appb-000011
Figure PCTCN2020085657-appb-000012
Figure PCTCN2020085657-appb-000013
Figure PCTCN2020085657-appb-000014
其中，在SEQ ID NO:3中，GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGA为第一HIV-1基因组序列(其中，GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG为截短的HIV-1 5’LTR(I型人免疫缺陷病毒5’端长末端重复序列long terminal repeat from human immunodeficiency virus-1)区序列，CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTC为病毒包装信号功能域ψ序列，)，GATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATT AACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC为第二HIV-1基因组序列(其中，AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTG为RRE功能域(Rev response element)序列)，GTTAAC为HpaI酶切位点，TTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAATTCAAAATTTT为第三HIV-1基因组序列，称为cPPT\CTS(central polypurine tract and central termin ation sequence)区序列，CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG为CMV(hunan cytomegalovirus，人巨细胞病毒)增强子区序列，GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC为CMV启动子区序列，ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACG CTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA为mCherry区序列，AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC为woodchuck hepatitis virus posttranscriptional regulatory element(WPRE)区序列，TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA G为第四HIV-1基因组序列，是缺失U3区域的HIV-1 3’LTR(3’长末端重复序列区序列)序列，AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA为SV40POLYA信号序列，TGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAAC为RSV(Rous sarcoma virus肉瘤病毒)启动子区序列。
根据本发明的实施例，相比于野生型pLV-mCherry，所述转移质粒的HIV-1 5’LTR区的5’端缺失2个GG。进而进一步提高了包装出病毒的滴度。
根据本发明的实施例，所述转移质粒具有SEQ ID NO:1或2所述的核苷酸序列。
在本发明的第四方面，本发明提出了一种病毒包装试剂盒。根据本发明的实施例，所述试剂盒包括前面所述的转移质粒。利用根据本发明实施例的试剂盒包装病毒，病毒的滴度显著提高。
根据本发明的实施例，所述试剂盒进一步包括辅助质粒。
在本发明的第五方面，本发明提出了一种包装病毒的方法。根据本发明的实施例，所述方法包括将病毒包装质粒对病毒包装工具细胞进行转染；将转染后的病毒包装工具细胞进行孵育，以便获得病毒，其中，所述病毒包装质粒包括前面所述的转移质粒和辅助质粒。根据本发明实施例的方法，显著提高了包装出的病毒的滴度。
根据本发明的实施例，所述病毒包装工具细胞为293T或293F。293T或293F方便培养，复制迅速，易于转染，进而进一步提高了病毒滴度。
需要说明的是，如无特殊说明，本申请所述的“转移质粒”是指携带待转导目的基因和HIV-1调控顺式元件序列信息的质粒，如pLV-mCherry，该质粒在病毒包装细胞中转录生成的RNA能够被包装进慢病毒载体中；本申请所述的“辅助质粒”是指除转移质粒外，携带HIV-1的gag/pol，rev或包膜蛋白序列信息的质粒，如psPAX2、pMDLg-pRRE、pRSV-rev或pMD2.G，该类质粒在病毒包装细胞中经转录、翻译后，以蛋白的形式参与到慢病毒载体的生命周期中。
附图说明
图1为根据本发明实施例的pLV-mCherry的质粒图谱；
图2为根据本发明实施例的pLV-mCherry转移质粒及其突变体模式图；
图3为根据本发明实施例的三质粒系统包装慢病毒滴度比较图；以及
图4为根据本发明实施例的四质粒系统包装慢病毒滴度比较图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，本申请所述的SEQ ID NO:1～4所示的转移质粒的序列是以HIV-1 5’LTR区的5’端为起点展示的序列，与SEQ ID NO:1～4具有同一性的转移质粒，均在本申请的保护范围内。同时，本领域技术人员所理解的是，质粒为环状结构，以任意位置的5’端为起点均可展示质粒的序列。以其它5’端为起点展示的转移质粒的序列，如果与SEQ ID NO:1～4仅是展示起点不同，但序列相同，或展示起点不同，但序列与SEQ ID NO:1～4具有同一性，也均在本申请的保护范围内。
需要说明的是，如无特别说明，本申请所述的“同一性”是指具有与本申请的序列具有至少70％、75％、80％、85％、90％、95％或99％同一性的序列。术语百分比(％)“序列同一性”描述了相对于构成模板核酸总长度，比对中的两个以上核酸序列中相一致的核苷酸的匹配(“命中”)数。换言之，通过使用比对，对于两个以上的序列或子序列，在对这些(子)序列进行比较和比对以在比较视窗或指定区域上获得用本领域已知的序列比较算法测得 的最大对应时，或者在进行人工比对和目视检查时，可以确定其相同的核苷酸的百分比
(例如，80％、85％、90％或95％的同一性)。此定义同样适用于测试序列的互补序列。本发明的目的在于提供三种新型生产高滴度的基于HIV-1的慢病毒载体的转移质粒。此三种质粒分别为：
一种新型生产高滴度的基于HIV-1的慢病毒载体的转移质粒(本申请中命名为△GG或pLV-△GG(1-2)-mCherry质粒)(序列如SEQ ID NO:4所示)，在慢病毒HIV-1基因组编码区的5’末端进行缺失突变，仅保留一个鸟苷酸，使得慢病毒生物滴度提高至原来的1.5～2倍。
一种新型生产高滴度的基于HIV-1的慢病毒载体的转移质粒(本申请中命名为Mut或pLV-Mut(1569-1577)-mCherry质粒)(序列如SEQ ID NO:1所示)，将慢病毒cPPT/CTS元件的5’端及5’端附近的9个碱基(GTTAACTTT)突变为8个碱基(ACTAGTTG)，使得慢病毒载体生物滴度提高至原来的1.3-1.5倍。
一种新型生产高滴度的基于HIV-1的慢病毒载体的转移质粒(本申请中命名为△GG&Mut或pLV-△GG(1-2)-Mut(1569-1577)-mCherry质粒)(序列如SEQ ID NO:2所示)，同时在慢病毒基因组编码区中引入两个突变，一是HIV-1基因组5’末端引入2个碱基的缺失突变，二是将cPPT/CTS元件的5’端及其附近的9个碱基(GTTAACTTT)突变为8个碱基(ACTAGTTG)，使得慢病毒生物滴度提高至原来的2-3倍。
Figure PCTCN2020085657-appb-000015
Figure PCTCN2020085657-appb-000016
Figure PCTCN2020085657-appb-000017
Figure PCTCN2020085657-appb-000018
Figure PCTCN2020085657-appb-000019
本发明获得的新型转移质粒相比于pLV-mCherry转移质粒，在生产慢病毒载体能力上具有先天优势，从根本上提高了慢病毒载体的产量。
△GG突变体中的突变是将文献报道中HIV-1的复制机理应用到慢病毒载体生产中，提高慢病载体产量。所使用的基于HIV-1的慢病毒载体保留了HIV-1基因组中的包装、逆转录和整合的顺式作用元件，用这些元件调控外源目的基因的转导。因此，病毒的包装、逆转录和整合方式仍同HIV-1一致。
HIV-1的基因组为正链RNA。研究表明，在HIV-1包装的过程中，全长的正链RNA可以单体形式用作信使RNA(mRNA)进行翻译，也可形成二聚体作为基因组RNA(gRNA)被包装进入病毒颗粒中。病毒通过调控单体和二聚体的比例，来调控病毒的复制周期进程。Siarhei等发现HIV-1全长RNA 5’端的鸟嘌呤数量为1-3个，单个鸟嘌呤RNA更加倾向于形成二聚体gRNA结构，被包裹入病毒颗粒中，形成成熟的病毒颗粒；而2个和3个鸟嘌呤的RNA则更倾向于作为mRNA招募核糖体等进行翻译。目前使用的基于HIV-1的慢病毒载体转移质粒中所编码的RNA起始位置均为3个鸟嘌呤，倾向于翻译，却不利于组装到病毒颗粒中。的确，慢病毒的包装过程中，会产生大量没有包装到gRNA的空壳病毒。但是，慢病毒载体包装的目的是产生更多的包装有gRNA的完整病毒颗粒，说明可以通过提高二聚体gRNA比例的方式来获得更多完整病毒颗粒，提高慢病毒初始产物的生物滴度。
Mut突变体是本申请的发明人在科学研究中意外发现地可以显著提高包装的慢病毒滴度的转移质粒。
△GG&Mut突变体是将上述两个突变同时引入慢病毒基因组中，两个突变可协同提高慢病毒滴度，获得的新型的生产高滴度的基于HIV-1的慢病毒载体的转移质粒。
下面将结合具体实施例对本发明进行进一步解释说明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从 商业途径得到。
实施例1
构建一种突变型转移质粒：含有GG(1-2)缺失突变的pLV-△GG(1-2)-mCherry质粒，此处△GG(1-2)表示转移质粒pLV-mCherry(简称WT，附图2A)中HIV基因组编码区第1至第2位碱基对缺失(附图2B)。
首先根据pLV-mCherry序列，设计overlapping PCR引物：
GG(1-2)-forward-primer-1:(5’-3’)
Figure PCTCN2020085657-appb-000020
GG(1-2)-forward-primer-2:(5’-3’)
Figure PCTCN2020085657-appb-000021
GG(1-2)-rerward-primer-1:(5’-3’)
Figure PCTCN2020085657-appb-000022
GG(1-2)-rerward-primer-2:(5’-3’)
Figure PCTCN2020085657-appb-000023
由基因合成服务公司合成上述序列。
分别以GG(1-2)-forward-primer-1和GG(1-2)-rerward-primer-2、GG(1-2)-forward-primer-2和GG(1-2)-rerward-primer-1为引物，以pLV-mCherry为模板，通过PCR方式获得产物片段A和B。回收片段A和B产物，再以片段A和B混合物为模板，GG(1-2)-forward-primer-1和GG(1-2)-rerward-primer-1为引物，通过over-lapping PCR方式获得产物片段C。通过酶切，将片段C连接至pLV-mCherry质粒载体的对应酶切位点之间，将该载体转化大肠杆菌stbl3感受态，经酶切和测序验证得到正确pLV-△GG(1-2)-mCherry质粒(简称△GG，如附图2B)。
实施例2
构建一种突变型转移质粒：含有1569-1577突变的pLV-Mut(1569-1577)-mCherry质粒，此处含有1569-1577突变表示将转移质粒pLV-mCherry中HIV基因组编码区第1569-1577位碱基对GTTAACTTT突变为ACTAGTTG(附图2C)。
首先根据pLV--mCherry序列，设计overlapping PCR引物：
Mut(1569-1577)-forward-primer-1:(5’-3’)
Figure PCTCN2020085657-appb-000024
Mut(1569-1577)-forward-primer-2:(5’-3’)
Figure PCTCN2020085657-appb-000025
Mut(1569-1577)-rerward-primer-1:(5’-3’)
Figure PCTCN2020085657-appb-000026
Mut(1569-1577)-rerward-primer-2:(5’-3’)
Figure PCTCN2020085657-appb-000027
由基因合成服务公司合成上述序列。
分别以Mut(1569-1577)-forward-primer-1和Mut(1569-1577)-rerward-primer-2、Mut(1569-1577)-forward-primer-2和Mut(1569-1577)-rerward-primer-1为引物，以pLV-mCherry为模板，通过PCR方式获得产物片段D和E。回收片段D和E产物，再以片段D和E混合物为模板，Mut(1569-1577)-forward-primer-1和Mut(1569-1577)-rerward-primer-1为引物，通过over-lapping PCR方式获得产物片段F。通过酶切，将片段F连接至pLV-mCherry质粒载体的对应酶切位点之间，将该载体转化大肠杆菌stbl3感受态，经酶切和测序验证得到正确pLV-Mut(1569-1577)-mCherry质粒(简称Mut，如附图2C)。
实施例3
构建一种突变型转移质粒：同时含有GG(1-2)缺失和1569-1577突变的pLV-△GG(1-2)-Mut(1569-1577)-mCherry质粒，此处△GG(1-2)表示转移质粒pLV-mCherry中HIV基因组编码区第1至第2位碱基对缺失，Mut(1569-1577)表示转移质粒pLV-mCherry中HIV基因组编码区第1569-1577位碱基对GTTAACTTT突变为ACTAGTTG(附图2D)。
取实施例1和实施例2中构建好的质粒，分别用SphI和NotI进行酶切，分别回收△GG的小片段和Mut的大片段。用T4连接酶将回收的两片段进行连接，转化大肠杆菌stbl3感受态，经酶切和测序验证得到正确pLV-△GG(1-2)-Mut(1569-1577)-mCherry质粒(简称△GG&Mut，如附图2D)。
实施例4
取实施例1和实施例2中构建好的质粒，以pLV-mCherry转移质粒为对照组，以psPAX2和pMD2.G为辅助质粒，用三质粒系统进行慢病毒包装。按转移质粒：psPAX2：pMD2.G＝2:1:1比例，用lipofect3000将质粒转染到293T细胞中，转染后50小时收集细胞培养物中病毒上清，经0.45μm滤头过滤后，进行滴度测定。
将293T细胞按3.6E+04cells/孔接种至24孔板内，培养过夜，以使细胞贴壁。通过10倍梯度稀释法，将过滤后的病毒上清进行稀释，稀释病毒液中添加8μg/mL的polybrene。弃293T孔内原培养基，加入病毒稀释液，室温，1000g，离心40分钟后，37℃、5％CO 2静置培养72h后，流式检测mCherry阳性率，计算对应样品滴度和同pLV-mCherry所获慢病毒滴度的比值。其中，数值>1则提高病毒滴度；数值<1，则降低病毒滴度；数值＝1，则无影响。结果如图3所示，Mut和△GG&Mut组获得的慢病毒的滴度相比于pLV-mCherry所获得的慢病毒滴度显著提高。
实施例5
取实施例1和实施例2中构建好的质粒，以pLV-mCherry转移质粒为对照组，以pMDLg-pRRE、pRSV-rev和pMD2.G为辅助质粒，用四质粒系统进行慢病毒包装。按转移质 粒：pMDLg-pRRE：pRSV-rev：pMD2.G＝1:1:1:1比例，用lipofect3000将质粒转染到293T细胞中，转染后50小时收集细胞培养上清，经0.45μm滤头过滤后，进行滴度测定。
将293T细胞按3.6E+04cells/孔接种至24孔板内，培养过夜，以使细胞贴壁。通过10倍梯度稀释法，将过滤后的病毒上清进行稀释，稀释病毒液中添加8μg/mL的polybrene。弃293T孔内原培养基，加入病毒稀释液，室温，1000g，离心40分钟后，37℃、5％CO 2静置培养72h后，流式检测mCherry阳性率，计算对应样品滴度和同pLV-mCherry所获慢病毒滴度的比值。其中，数值>1则提高病毒滴度；数值<1，则降低病毒滴度；数值＝1，则无影响。结果如图4所示，Mut和△GG&Mut组获得的慢病毒的滴度相比于pLV-mCherry所获得的慢病毒滴度显著提高。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (14)

  1. 一种分离的核酸，其特征在于，具有X 1X 2TAX 5X 6X 7TX 9所示的核苷酸序列，
    其中，X 1为A或G；X 2为T或C；X 5为A或G；X 6为C或T；X 7为T或缺失；X 9为T或G；但不具有GTTAACTTT所示的核苷酸序列。
  2. 根据权利要求1所述的核酸，其特征在于，具有ACTAGTTG所示的核苷酸序列。
  3. 一种转移质粒，其特征在于，携带权利要求1或2所述的核酸。
  4. 根据权利要求3所述的转移质粒，其特征在于，所述核酸位于所述转移质粒中cPPT/CTS元件的5’端及5’端上游。
  5. 根据权利要求4所述的转移质粒，其特征在于，所述核酸位于所述转移质粒中cPPT/CTS元件的5’端2～5bp及5’端上游4～8bp范围内，优选地，所述核酸位于所述转移质粒中cPPT/CTS元件的5’端3bp及5’端上游6bp范围内。
  6. 根据权利要求5所述的转移质粒，其特征在于，所述转移质粒具有SEQ ID NO:1所示的核苷酸序列。
  7. 根据权利要求3所述的转移质粒，其特征在于，所述转移质粒包含启动子区和HIV-1 5’LTR区，所述启动子区与HIV-1 5’LTR区可操作地连接，所述HIV-1 5’LTR区的5’端具有单个鸟甘酸，优选地，所述启动子为RSV启动子。
  8. 根据权利要求7所述的转移质粒，其特征在于，所述转移质粒具有SEQ ID NO:2所示的核苷酸序列。
  9. 一种转移质粒，其特征在于，相比于野生型pLV-mCherry，具有下列突变的至少之一，
    第1569位碱基由G突变为A；
    第1570位碱基由T突变为C；
    第1573位碱基由A突变为G；
    第1574位碱基由C突变为T；
    第1575位碱基缺失；
    第1577位碱基由T突变为G。
  10. 根据权利要求9所述的转移质粒，其特征在于，相比于野生型pLV-mCherry，所述转移质粒的HIV-1 5’LTR区的5’端缺失2个GG。
  11. 根据权利要求9或10所述的转移质粒，其特征在于，所述转移质粒具有SEQ ID NO:1或2所述的核苷酸序列。
  12. 一种病毒包装试剂盒，其特征在于，包括权利要求3～11任一项所述的转移质粒；任选地，进一步包括辅助质粒。
  13. 一种包装病毒的方法，其特征在于，将病毒包装质粒对病毒包装工具细胞进行转染；将转染后的病毒包装工具细胞进行孵育，以便获得病毒，
    其中，所述病毒包装质粒包括权利要求3～11任一项所述的转移质粒和辅助质粒。
  14. 根据权利要求13所述的方法，其特征在于，所述病毒包装工具细胞为293T或293F。
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