CN114478713B

CN114478713B - CMV envelope protein packaging lentiviral vector and application thereof

Info

Publication number: CN114478713B
Application number: CN202210157391.2A
Authority: CN
Inventors: 沈健; 葛永; 刘正明; 杨淑青
Original assignee: Jiangsu Mengbili Biological Technology Co ltd
Current assignee: Jiangsu Mengbili Biological Technology Co ltd
Priority date: 2022-02-21
Filing date: 2022-02-21
Publication date: 2023-03-28
Anticipated expiration: 2042-02-21
Also published as: CN114478713A

Abstract

The invention provides a cytomegalovirus CMV envelope protein packaging lentiviral vector and an application thereof, belonging to the field of biomedical engineering. Aiming at the limitation of the prior lentivirus vector transfection NK cell, the invention transfers cytomegalovirus envelope proteins (gH, gL and gO) into a packaging cell 293T, and packages lentivirus DNA to generate virus. The invention replaces VSV-G with CMV membrane protein (gH/gL/gO) to pack lentivirus pseudovirus, not only maintains higher virus titer, has stable transfection function to epithelial cells, but also can effectively identify NK cells and mediate virus to enter cells, thereby realizing the purpose of efficiently transfecting the NK cells, providing a new tool for gene therapy and cell transfection research, and being applied to large-scale research and production.

Description

CMV envelope protein packaging lentiviral vector and application thereof

Technical Field

The invention relates to the field of biomedical engineering, in particular to a cytomegalovirus CMV envelope protein packaging lentiviral vector and an application thereof.

Background

Lentiviral Vector (LV) refers to a viral vector derived from human immunodeficiency virus-1 (HIV-1), and the Lentiviral vector contains genetic information required for packaging, transfection and stable integration, and is a main component of a Lentiviral vector system. Under the assistance of lentivirus packaging plasmid and cell line, the lentivirus vector carrying exogenous gene is virus packaged into virus particle with infectivity, and the expression of exogenous gene in cell or living tissue is realized through infecting cell or living tissue. Lentiviral vector helper components include: lentivirus packaging plasmids and cell lines that produce viral particles. The lentivirus vector contains genetic information required by packaging, transfection and stable integration, can effectively infect various cells, can be a non-dividing cell, becomes an effective vector for exogenous gene transfer at present, and is widely applied to the field of gene therapy, particularly CAR-T therapy.

Viruses recognize and enter target cells through Glycoproteins (GP) on their surface, which makes the virus selective for infecting cells. Clinical studies have found that Human Herpes Virus (HHV) not only infects lymphocytes, but also NK cells. The human herpesvirus comprises three types of alpha, beta and gamma, wherein the alpha type comprises HSV-1 (HHV 1), HSV-2 (HHV 2) and VZV (HHV 3), and the host range of the human herpesvirus is wide and mainly latent in nerve cells; betatyping includes HCMV (HHV 5), HHV6 and HHV7, which have a narrow host range and are predominantly latent in lympho-reticuloendothelial cells; gamma typing includes EBV (HHV 4) and HHV8, which have the narrowest host range and are predominantly latent in B lymphocytes. Wherein Human Cytomegalovirus (HCMV) belongs to Human herpesvirus 5 type, is known as lymphotropic cell and can effectively remain latent in NK cell and reticuloendothelial cell.

Currently, there is a technology for preparing a vector using human cytomegalovirus CMV. For example, patent CN1482239A discloses a recombinant human herpes simplex virus type 1 "recombinant HSV1-Lenti-heiper virus" which utilizes the expression cassettes gag, pol, VSV-G, rev for various trans proteins required for HSV1 expression lentiviral vector packaging for packaging lentiviruses for transfection, which remains essentially a VSV-G packaged lentivirus. Patent CN110036112A discloses CMV vectors lacking active UL128, UL130, UL146 and UL147 proteins, which may also contain one or more Microrna Regulatory Elements (MREs) that limit CMV expression. Immunization with the disclosed CMV vectors allows the selection of different CD8+ T cell responses-CD 8+ T cells restricted by MHC-Ia, MHC-II or MHC-E.

CAR-T therapy is a Chimeric Antigen Receptor T-Cell Immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy) that recognizes and attacks tumor cells through specific receptors. This function of CAR-T relies primarily on cytotoxic T cells (Tc), which, when activated, release granzymes and perforin, inducing apoptosis in the target cell. In addition to the cytotoxic effect of Tc in humans, natural Killer (NK) cells have similar functions. However, NK cells are different from T cells and B cells, and are lymphocytes which can kill tumor cells and virus infected cells in a non-specific way without pre-sensitization, and the treatment of tumors by modifying NK cells by using biotechnology is a promising therapy.

At present, the CAR-T is prepared by using VSV-G enveloped pseudovirus as a vector, namely a lentiviral vector commercialized at present. LV enveloped by VSV-G was able to transfect T lymphocytes efficiently, but not NK cells efficiently. Therefore, it is highly desirable to provide a lentiviral vector for the purpose of efficiently transfecting NK cells.

Disclosure of Invention

Aiming at the defects, the invention provides a cytomegalovirus CMV envelope protein packaging lentivirus vector. Aiming at the limitation of the transfection of NK cells by the conventional lentiviral vector, the cytomegalovirus envelope proteins (gH, gL and gO) are transferred into a packaging cell 293T, and the lentivirus DNA is packaged to generate virus. The cytomegalovirus envelope protein not only can identify NK cells, but also can mediate viruses to enter the cells so as to realize the purpose of efficiently transfecting the NK cells.

In order to achieve the above object, the technical solution of the present invention is as follows:

in one aspect, the invention provides a cytomegalovirus envelope protein comprising envelope proteins (glycoprotens H, L and O, i.e., gH, gL and gO).

Specifically, the coding sequence of the envelope protein gH is a nucleotide sequence shown by SEQ ID NO. 1, the coding sequence of the envelope protein gL is a nucleotide sequence shown by SEQ ID NO. 2, and the coding sequence of the envelope protein gO is a nucleotide sequence shown by SEQ ID NO. 3.

Specifically, cytomegalovirus recognizes target cells and enters cells as determined by envelope proteins gH, gL, and gO.

In another aspect, the invention provides the use of the cytomegalovirus envelope protein in the construction of lentiviral vectors.

In still another aspect, the present invention provides a lentiviral vector comprising the cytomegalovirus envelope proteins gH, gL and gO as described above.

Specifically, the lentiviral vector comprises a gL/gO plasmid and a gH plasmid.

More specifically, the gL/gO plasmid further comprises a T2A (2A self-clearing peptides) peptide, wherein the coding sequence of the T2A peptide is the nucleotide sequence shown in SEQ ID NO. 4

More specifically, the nucleotide sequences of gL and gO are connected through the nucleotide sequence of T2A peptide to form a coexpression structure.

In another aspect, the invention provides the use of the above cytomegalovirus envelope protein or lentiviral vector for the preparation of a lentiviral pseudovirus.

In another aspect, the present invention provides a method for preparing a lentivirus pseudovirus, comprising the steps of:

(1) Connecting the nucleotide sequences of gL and gO through the nucleotide sequence of T2A peptide to form a co-expression structure, cloning the co-expression structure into an expression vector, transfecting cells, and screening packaging cells stably expressing gL and gO by antibiotics;

(2) And (2) independently cloning gH into an expression vector, transferring the gH together with Plenti, pLP1 and pLP2 into packaging cells which are obtained by screening in the step (1) and stably express gL and gO, and culturing the cells to generate the lentivirus pseudovirus with cytomegalovirus membrane protein.

Specifically, the cytomegalovirus envelope proteins gH and gL can form a dimer, which are conserved proteins in the herpesvirus family and are essential structures for cell fusion. Coexpression of gH and gL in the cell promotes cell fusion and syncytia formation, and gO does not play a role in the above phenomenon, but gO is a membrane glycoprotein essential for virus entry into the cell, and binding of gH, gL formed trimer to specific proteins on the surface of target cells mediates entry of cytomegalovirus into the cell. Because of the adverse effects that fusion and syncytia formation of cells by gH and gL may have on the packaging cells, gL/gO and gH were transferred in batches into packaging cells, respectively.

Specifically, the T2A peptide in step (1) is derived from the virus of the phaedomyza sativae (thosa asigna virus), and such peptides have a sequence motif, which results in the ribosome not being able to link at the junction of the last glycine (G) and proline (P), thereby causing a "shearing" effect, which releases the synthesized peptide chain in the first half early, and thus forms two peptides bounded by the 2A peptide. And simultaneously, a GSG amino acid coding sequence is designed and added in front of the T2A sequence, so that the shearing efficiency is further improved.

Specifically, the expression vector in the step (1) is an MSCV (mouse stem cell virus) vector.

Specifically, the transfected cells in step (1) are 293-T cells.

Specifically, the antibiotic in the step (1) is bleomycin. In certain embodiments of the invention, an IRES (Internal ribosome entry site) is used to link the sequence of the antibiotic resistance gene (bleomycin resistance gene BleoR) after the gL-T2A-gO sequence to facilitate selection of stably expressing cell lines.

More specifically, the bleomycin resistance gene BleoR has a nucleotide sequence shown in SEQ ID NO. 5.

Specifically, the expression vector in the step (2) is pcDNA3.1+. In the present invention, after gH was cloned into pcDNA3.1+ alone, the G glycoprotein of vesicular stomatitis virus (VSV-G), which is a fusogenic coat protein, in a conventional lentiviral 4 plasmid system (e.g., invitrogen) was replaced by this plasmid.

Specifically, pLenti, pLP1 and pLP2 described in step (2) are the other 3 plasmids of the traditional lentiviral 4 plasmid system, wherein pLenti is a lentiviral vector expression plasmid and pLP1 and pLP2 encode gag, pol and Rev genes, respectively. Wherein, GAG encodes a p55 protein precursor consisting of about 500 amino acids, which is cleaved by proteases to form the nucleocapsid protein (p 7), the inner membrane protein (p 17) and the capsid protein (p 24) of the virus; POL encodes polymerase precursor protein, form protease, integrase, reverse transcriptase, ribonuclease H after cutting; REV encodes a protein that regulates the expression of HIV virion proteins and serves the primary function of promoting the conversion of HIV gene expression from early (transcription of regulatory protein mRNA) to late (transcription of HIV structural protein mRNA).

Specifically, in step (2), when three plasmids of gH, pLenti, pLP1 and pLP2 are transferred into the packaging cells which are obtained by screening and stably express gL and gO together, the vector expression plasmids are transcribed to generate lentivirus genome mRNA, rev protein is combined with RRE in the lentivirus genome mRNA, and the genome mRNA is transferred from nucleus to cytoplasm. The genomic mRNA is assembled with capsid proteins, reverse transcriptase, integrase, CMV envelope proteins (gH/gL/gO will form trimers within the cell), etc. into pseudoviral particles, producing a lentiviral pseudovirus with cytomegalovirus membrane proteins.

In still another aspect, the present invention provides the use of the above cytomegalovirus envelope protein, lentiviral vector and/or lentiviral pseudovirus for transfecting NK cells.

Compared with the prior art, the invention has the positive and beneficial effects that:

the invention replaces VSV-G with CMV membrane protein (gH/gL/gO) to pack lentivirus pseudovirus, not only maintains higher virus titer, has stable transfection function to epithelial cells, but also can effectively identify NK cells and mediate virus to enter the cells, thereby realizing the purpose of efficiently transfecting the NK cells. The slow virus pseudovirus provides a new tool for gene therapy and cell transfection research, and can be applied to large-scale research and production.

Drawings

FIG. 1 is a schematic representation of cloning of gL and gO into MSCV vectors.

FIG. 2 is a schematic diagram of the cloning of gH into pcDNA3.1+ vector.

FIG. 3 is a flow chart of a conventional pseudovirus packaging process.

FIG. 4 is a flow chart of pseudovirus packaging according to the present invention.

FIG. 5 is a graph showing the results of the flow assay in Experimental example 2.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

The examples, where no specific techniques or conditions are indicated, are carried out according to the techniques or conditions described in the literature of the art (for example, see J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, ed. By Huang Pe, et al) or according to the instructions of the product.

Example 1: plasmid vector construction

The Snapgene software was used to analyze the restriction sites of all cloned DNA, and then CMV-G and other sequences were inserted into the DNA to design and construct a complete new expression plasmid using conventional molecular cloning techniques in the art. Each gene sequence was amplified by high Fidelity PCR (Phanta Super-Fidelity DNA polymers, vazyme, nanjing) or obtained by digestion of plasmid. The amplification primers were synthesized by hong Xue company (see Table 1 below), and the PCR reaction system was shown in Table 2 below.

TABLE 1 primers used for vector construction

TABLE 2 PCR reaction System

Reagent	Add volume (μ L)
		5×Buffer	10
5xdNTP	4
		10nM F primer	2
10nM R primer	2
		Phanta Polymeras(Vazyme)	0.5
Total volume	50

1. MSCV-IRES-BleoR vector construction

1. The BleoR fragment was generated by PCR using pTracer-CMV2 (Invitrogen) as a template. Specific primer sequences are detailed in Table 1 above, wherein the 5 'primer (BleoR-F) has an NcoI site and the 3' primer (BleoR-R) has an SalI site, and PCR amplification was performed according to the reaction system of Table 2 above and the reaction procedure of Table 3 below. The amplified BleoR fragment was cloned into the MSCV vector MIGR1 (university of Pennsylvania), and the MSCV-IRES-BleoR construction was performed in place of EGFR. Specifically, the MIGR1 vector and the BleoR PCR product were cut with the corresponding restriction enzymes (New England Labotory, USA), gel-separated, and then recovered and purified by agarose gel DNA recovery kit (Beijing Jiang Co., ltd.) according to the method. Ligation into circular plasmids was carried out by T4 DNA ligase (see Table 4). Finally, the vector is sent to sequencing to confirm that the construction of the vector is successful

TABLE 3 PCR reaction procedure

TABLE 4T 4 Ligase ligation, overnight at 14 ℃

2. Construction of gL/gO expression plasmid

Genomic DNA of CMV is derived from clinical peripheral blood specimens (CMV-infected individuals) and PCR is performed to generate gL and gO gene DNA fragments, the specific primer sequences are detailed in Table 1 above, and the PCR reaction system is detailed in Table 2 above.

(1) gL gene fragment: the 5 'primer (CMVgL-F) has a BgII cleavage site and the 3' primer (CMVgL-R) has a T2A sequence so as to fuse with the gO gene fragment to form a complete reading frame. The PCR reaction procedure is detailed in Table 5 below.

TABLE 5 PCR reaction procedure

(2) gO gene fragment: the 5' primer (CMVgO-F) carries a T2A sequence complementary to the 3' sequence of the gL gene PCR product of step (1), and the 3' primer (CMVgO-R) carries an XhOI site. The PCR reaction procedure is detailed in Table 6 below.

TABLE 6 PCR reaction procedure

(3) gL/gO of T2A linkage: the gL and gO gene fragments generated by the PCR in the above steps (1) and (2) were fused and extended by complementary T2A sequence under the action of DNA polymerase (Vazyme, see above, nanjing) to obtain full-length DNA (see Table 7 below for details of reaction procedure). Then, using the diluted product as a template, a gL-T2A-gO fragment was obtained by further amplification using a gL gene 5 'primer (CMVgL-F) and a gO gene 3' primer (CMVgO-R) (see Table 8 below for details of the reaction procedure).

TABLE 7 PCR reaction procedure

TABLE 8 PCR reaction procedure

(4) Cloning of the gL-T2A-gO fragment into the MSCV-IRES-Bleo vector: and (4) reacting the gL-T2A-gOPCR fragment obtained by amplification in the step (3) with a corresponding restriction enzyme overnight, and identifying and separating the enzyme-cleaved fragment by gel electrophoresis and recovering DNA (the method is the same as the method). Ligation was performed by using T4 DNA ligase to obtain circular plasmids (the reaction system is shown in Table 4). The ligation product is transformed into Stale Escherichia coli competent cells (New England Lab, NEB), and the clone is screened by ampicillin, and then further sequencing verifies that the plasmid construction is successful, and finally the lentivirus basic plasmid containing gL-T2A-gO is obtained (see the attached figure 1 for details).

3. Construction of gH Gene expression plasmid

(1) generation of gH fragments: PCR amplification was performed using CMV genomic DNA (see above) as a template, and the details of the PCR reaction system are shown in Table 2 above. The 5' primer (CMVgH-F) has an EcoRI site; the 3' primer (CMVgH-R) carries an XhoI site.

TABLE 9 PCR reaction procedure

(2) The gH gene obtained by amplification in the step (1) is cloned into pcDNA3.1+ vector after enzyme digestion and connection to obtain pgH plasmid (see the attached figure 2 for details).

4. Preparation of gL/gO-expressing packaging cells

And (3) transfecting 293T cells with the lentivirus basic plasmid containing gL-T2A-gO prepared in the second step by using a Lipo3000 transfection reagent as a medium, carrying out passage on the cells after three days, adding Zeocin (150 mu g/mL) for screening, and obtaining 293T packaging cells for stably expressing gL/gO protein after 3 weeks.

Example 2: virus package

The lentiviral vector used was pLL3.7 (Addgene), which contained eGFP and was used as a marker for the success or failure of transfection. The traditional pseudovirus packaging process is shown in figure 3, and the pseudovirus packaging process of the invention is shown in figure 4. The traditional lentivirus system (i.e., VSV-G membrane protein packaging) pLL3.7 with packaging plasmids (pLP 1, pLP2, pLP-VSVG) transfected 293T cells; in the present invention, the slow virus system (CMV-G package) is used to transfect pLL3.7 and packaging plasmids (pLP 1, pLP2, pgH) into 293T packaging cells expressing gL/gO.

The specific procedures were according to the Invitrogen instructions, during virus packaging: packaging plasmid 9. Mu.g, vector plasmid 3. Mu.g, CPT high efficiency transfection reagent purchased from Wuhanweinaoxi Biotech. Inoculation 5X 10 one day before transfection ⁶ 293T cells/100 mm dish, 5% CO at 37% ₂ The culture was carried out overnight in an incubator. The medium was changed to non-resistant complete medium 3-4h before transfection the next day. Two sterile, clean centrifuge tubes, labeled a and B: adding a proper amount of sterile water, plasmid DNA and 50 mu L of buffer B (the total amount is 500 mu L) into a tube B, uniformly mixing, and slowly dropwise adding the mixture into a tube A containing 500 mu L of buffer A; the transfection mixture was mixed by bubbling with a suction nozzle and allowed to stand at room temperature for 30min. Adding the transfection mixture dropwise to the cell culture plate, gently shaking the culture dish to spread the transfection mixture uniformly over the entire plate, mixing, adding to 37 deg.C, 5% CO ₂ And changing the culture solution after culturing for 4-6h in the incubator. Changing the culture broth to fresh complete medium containing the diabody on the third day, at 37 deg.C, 5% ₂ Culturing in the incubator for 24-48 hr, collecting virus supernatant, and storing at-80 deg.C.

Experimental example 1: viral titer assay

After the 293T cells in good growth state were digested and counted, the cells were plated in 24-well plates at 1X 10 cells per well ⁵ And (4) cells. The next day, the virus was diluted in 10-fold gradient and the cells prepared above were placed after 5 serial dilutions. On the third day, the culture medium was added, and 500. Mu.L of complete culture medium was added to each well. On the fifth day, cells were collected and the proportion of cells expressing GFP positive was determined by flow cytometry loading. Viral titer (TU/mL) = (10) ⁵ Number of cells × GFP +%) × 1000 × dilution.

Calculating the virus titer, wherein the titer of the lentivirus CMV-G pseudovirus is 6.8 multiplied by 10 ⁶ TU/mL, titer of conventional lentivirus VSV-G pseudovirus 8X 10 ⁶ TU/mL, indicating that the transfection efficiency of the CMV-G pseudovirus is obviously better than that of the VSV-G pseudovirus.

Experimental example 2: NK cell transfection assay

1. NK cell isolation and culture

Mononuclear cells in human peripheral blood were separated from lymphocyte separation medium, and then NK cells (CD 3-CD56 +) were separated from PBMC using immunomagnetic beads (Dynal NK cell isolation kit), and the purity of the separation was checked by flow cytometry. The details of the culture method of NK cells are described in patent CN112852728A, example 1.

2. NK cell transfection efficiency detection

NK cells were counted and seeded in 24-well plates at 5X 10 per well ⁴ (ii) individual cells; the 24-well plate was previously coated with recombinant human Fibronectin (rh-FN) (Beijing Boerci technologies, inc.) (5. Mu.g/cm for Fibronectin) ² Standing at room temperature for 30-40 min). The next day, the culture solution is changed, 500 μ L of fresh culture solution is added with concentrated lentivirus, polybrene (polybrene) is added to the final concentration of 8 μ g/mL, and cells are infected after uniform mixing; 6-8h later, 100. Mu.L of fresh culture medium was supplemented, at 37 ℃ C. And 5% CO ₂ Culturing in an incubator overnight; on the third day, cells were collected and washed twice with PBS, and cells were transferred to 12-well plates for culture; when the cells in the 12-pore plate grow more than 90%, collecting the cells for flow cytometry analysis, and obtaining the number of fluorescence positive cells. The transfection efficiency of the pseudovirus was judged by comparing the number of fluorescence positive cells with the control.

The detection result is shown in FIG. 5, wherein the lentivirus CMV-G pseudovirus shows stronger transfection effect, the transfection is positive by 26.4%, and the transfection is positive by 17.1% for VSV-G pseudovirus.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Jiangsu Monpeteli Biotech Co., ltd

<120> CMV envelope protein packaging lentiviral vector and application thereof

<130> 20210716

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 2232

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

atgcggcccg gcctcccctt ctacctcacc gtcttcgccg tctacctcct cagtcaccta 60

ccctcgcaac gatatggcgc agacgccgca tccgaagcgc tggaccctca cgcgtttcac 120

ctactactca acacctacgg gagacccatc cgtttcctgc gtgaaaacac cacccagtgc 180

acctacaaca gcagcctccg taacagcacg gtcgtcaggg aaaacgccat cagtttcaac 240

tttttccaaa gctataatca atactatgta ttccatatgc ctcgatgtct ttttgcgggt 300

cctctggcgg agcagtttct gaaccaggta gatctgaccg aaaccctaga aagataccaa 360

cagagactta acacctacgc attggtatcc aaagacctgg ctagctaccg atctttttcg 420

cagcagctga aggcacaaga cagcctgggt cagcagccca ccaccgtgcc accgcccatt 480

gatctgtcaa tacctcacgt ctggatgcca ccccaaacca ctccacacga ctggaaggga 540

tcgcacacca cctcgggact acatcggcca cactttaacc agacctgtat cctctttgat 600

ggacacgatc tgcttttcag caccgttacg ccctgtctgc accagggctt ttacctcatg 660

gacgaactac gttacgttaa aatcacactg accgaggact tcttcgtagt tacggtatcc 720

atagacgacg acacacccat gctgcttatc ttcggtcatc ttccacgcgt actcttcaaa 780

gcgccctatc aacgcgacaa ctttatacta cgacaaactg aaaaacacga gctcctggta 840

ctagttaaga aaactcaact gaaccgtcac tcctatctca aagactcgga ctttctcgac 900

gccgcactcg acttcaacta cctggacctc agcgcactgc tacgtaacag ctttcaccgt 960

tacgctgtag acgtactcaa aagcggtcga tgtcaaatgc tggaccgccg cacggtagaa 1020

atggcctttg cctacgcatt agcactgttc gcggcagccc gacaagaaga ggccggcacc 1080

gaaatctcca tcccacgggc cctagaccgc caggccgcac tcttacaaat acaagaattt 1140

atgatcacct gcctctcaca aacaccacca cgcaccacat tgctgctata tcccacggcc 1200

gtggacctgg ccaaacgagc cctctggacg ccggaccaga tcaccgacat caccagcctc 1260

gtacgcctgg tctacatact ttctaaacag aatcagcaac atctcattcc ccagtgggca 1320

ctacgacaga tcgccgactt tgccctacaa ttacacaaaa cgcacctggc ctcttttctt 1380

tcagccttcg cgcgccaaga actctacctc atgggcagcc tcgtccactc catgctggta 1440

catacgacgg agagacgcga aatcttcatc gtagaaacgg gcctctgttc attggccgag 1500

ctatcacact ttacgcagtt gctagctcat ccgcaccacg aatacctcag cgacctgtac 1560

acaccctgtt ccagtagcgg gcgacgcgat cactcgctcg aacgccttac gcgtctcttc 1620

cccgatgcca ccgtccccgc taccgttccc gccgccctct ccatcctatc taccatgcaa 1680

ccaagcacgc tggaaacctt ccccgacctg ttctgtctgc cgctcggcga atccttctcc 1740

gcgctgaccg tctccgaaca cgtcagttat gtcgtaacaa accagtacct gatcaaaggt 1800

atctcctacc ctgtctccac caccgtcgta ggccagagcc tcatcatcac ccagacggac 1860

agtcaaacta aatgcgaact gacgcgcaac atgcatacta cacacagcat tacagcggcg 1920

ctcaacattt cgctagaaaa ctgcgccttt tgccaaagcg ccctgctaga atacgacgac 1980

acgcaaggcg tcatcaacat catgtacatg cacgactcgg acgacgtcct tttcgccctg 2040

gatccctaca atgaagtggt ggtctcatct ccacgaactc actacctcat gcttttgaaa 2100

aacggtacgg tcctggaagt aactgacgtc gtcgtggacg ccaccgacag ccgtctcctc 2160

atgatgtccg tctacgcgct gtcggccatc atcggcatct atctgctcta ccgcatgctc 2220

aagacatgct ga 2232

<210> 2

<211> 2294

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

atgtgccgcc gcccggattg cggcttctct ttctcacctg gaccggtgat actgctgtgg 60

tgttgccttc tgctgtccat tgtttcctca gccgccgtca gcgtcgctcc taccgccgcc 120

gagaaagtcc ccgcggagtg ccccgaacta acgcgccgat gcctgttggg tgaggtgttt 180

cagggtgaca agtatgaaag ttggctgcgc ccgttggtga atgttaccgg gcgcgatggc 240

ccgctatcgc aacttatccg ttaccgtccc gttacgccgg aggccgccaa ctccgtgctg 300

ttggacgatg ctttcctgga cactctggcc ctgctgtaca acaatccgga tcaattgcgg 360

gccctgctga cgctgttaag ctcggacaca gcgccgcgct ggatgacggt aatgcgcggc 420

tacagcgagt gcggcgatgg ctcgccggcc gtgtacacgt gcgtggacga cctgtgccgc 480

ggctacgacc tcacgcgact gtcatacggg cgcagcatct tcacggaaca cgtgttaggc 540

ttcgagctgg tgccaccgtc tctctttaac gtggtggtgg ccatacgtaa cgaagccacg 600

cgtaccaacc gcgccgtgcg tctgcccgtg agcaccgctg ccgcgcccga gggcatcacg 660

ctcttttacg gcctgtacaa cgcggtgaag gaattctgcc tgcgtcacca gctggacccg 720

ccgctgctac gccacctaga taaatactac gccggactgc cgcccgagct gaagcagacg 780

cgcgtcaacc tgccggctca ctcgcgctat ggccctcaag cagtggatgc tcgcggttcg 840

ggggaaggac gcggttcgct gttgacctgc ggtgatgtag aagaaaatcc tggtccaatg 900

gggagaaaag agatgatggt gagagacgtc cctaagatgg tgtttctaat atctatatct 960

ttcttgcttg tttctctcat aaactgtaaa gttatgtcaa aagcgcttta taatcgtcct 1020

tggaggggct tggtactgtc taagataggc aaatataaat tagatcagct taagttagaa 1080

attttgagac aactagaaac gactatttct acaaaataca atgtaagtaa acaaccggtt 1140

aaaaatctca ctatgaacat gacaaagttt ccacaatact acattttagc gggccccatt 1200

cagaattata gtataaccta tctgtggttt gatttttata gtacccagct tagaaaaccc 1260

gcaaaatacg tttactcaca gtacaatcat acggctaaaa cgataacatt cagaccccca 1320

ccttgtggta ctgtgccttc catgacttgt ctttccgaaa tgctaaacgt ttccaaacgt 1380

aatgatactg gcgaacaagg ttgcggtaat ttcaccacgt tcaaccccat gtttttcaat 1440

gtaccgcgtt ggaacaccaa attgtacgtg ggtccgacta aggttaacgt agatagtcaa 1500

acgatttatt ttctaggttt aaccgccctg cttttacgtt acgcacaacg caactgtaca 1560

cacagtttct acctggttaa cgccatgagc cggaatctat ttcgcgtccc caagtatatt 1620

aacggcacca agttaaaaaa cactatgcga aaactaaaac gtaaacaagc gcccgttaag 1680

gaacaattcg aaaaaaaagt taagaaaact cagagtacta ctacgccata cttttcctat 1740

acaacgtctg ccgctctcaa cgtcactact aacgtgactt atagtattac taccgccgca 1800

aggcgggttt ccacgtctac aattgcttat cgtcctgata gcagctttat gaagtccatt 1860

atggccacac agttaaggga cctagcaacg tgggtgtata ccactctacg ttaccggcaa 1920

aatccttttt gtgaaccaag ccgcaaccga accgccgtgt cagaatttat gaaaaacacg 1980

cacgtactaa tccgtaacga aacgccgtac actatttacg gtactctcga catgagctcc 2040

ttatattaca acgaaaccat gttcgtggaa aacaaaacag cttccgatag taacaaaact 2100

acacctacgt caccatcaat ggggtttcag agaacattta tagatcccct gtgggactat 2160

ctagactcgc tgctgtttct agatgagatt cgtaacttta gcctccggtc acccacgtat 2220

gtaaacctta ccccgccgga acaccgccgg gctgtaaatc tgtccaccct caatagcctt 2280

tggtggtggt tgca 2294

<210> 3

<211> 1401

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

atggggagaa aagagatgat ggtgagagac gtccctaaga tggtgtttct aatatctata 60

tctttcttgc ttgtttctct cataaactgt aaagttatgt caaaagcgct ttataatcgt 120

ccttggaggg gcttggtact gtctaagata ggcaaatata aattagatca gcttaagtta 180

gaaattttga gacaactaga aacgactatt tctacaaaat acaatgtaag taaacaaccg 240

gttaaaaatc tcactatgaa catgacaaag tttccacaat actacatttt agcgggcccc 300

attcagaatt atagtataac ctatctgtgg tttgattttt atagtaccca gcttagaaaa 360

cccgcaaaat acgtttactc acagtacaat catacggcta aaacgataac attcagaccc 420

ccaccttgtg gtactgtgcc ttccatgact tgtctttccg aaatgctaaa cgtttccaaa 480

cgtaatgata ctggcgaaca aggttgcggt aatttcacca cgttcaaccc catgtttttc 540

aatgtaccgc gttggaacac caaattgtac gtgggtccga ctaaggttaa cgtagatagt 600

caaacgattt attttctagg tttaaccgcc ctgcttttac gttacgcaca acgcaactgt 660

acacacagtt tctacctggt taacgccatg agccggaatc tatttcgcgt ccccaagtat 720

attaacggca ccaagttaaa aaacactatg cgaaaactaa aacgtaaaca agcgcccgtt 780

aaggaacaat tcgaaaaaaa agttaagaaa actcagagta ctactacgcc atacttttcc 840

tatacaacgt ctgccgctct caacgtcact actaacgtga cttatagtat tactaccgcc 900

gcaaggcggg tttccacgtc tacaattgct tatcgtcctg atagcagctt tatgaagtcc 960

attatggcca cacagttaag ggacctagca acgtgggtgt ataccactct acgttaccgg 1020

caaaatcctt tttgtgaacc aagccgcaac cgaaccgccg tgtcagaatt tatgaaaaac 1080

acgcacgtac taatccgtaa cgaaacgccg tacactattt acggtactct cgacatgagc 1140

tccttatatt acaacgaaac catgttcgtg gaaaacaaaa cagcttccga tagtaacaaa 1200

actacaccta cgtcaccatc aatggggttt cagagaacat ttatagatcc cctgtgggac 1260

tatctagact cgctgctgtt tctagatgag attcgtaact ttagcctccg gtcacccacg 1320

tatgtaaacc ttaccccgcc ggaacaccgc cgggctgtaa atctgtccac cctcaatagc 1380

ctttggtggt ggttgcagta a 1401

<210> 4

<211> 63

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

ggttcggggg aaggacgcgg ttcgctgttg acctgcggtg atgtagaaga aaatcctggt 60

cca 63

<210> 5

<211> 377

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

atggatgcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg 60

gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc 120

ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg 180

gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg 240

gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag 300

cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg 360

gccgaggagc aggactg 377

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

ccgccatgga tgccaagttg acc 23

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

cccgtcgact cagtcctgct cctcg 25

<210> 8

<211> 32

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

gaattagatc tatgtgccgc cgcccggatt gc 32

<210> 9

<211> 84

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

gattttcttc tacatcaccg caggtcaaca gcgaaccgcg tccttccccc gaaccgcgag 60

catccactgc ttgagggcca tagc 84

<210> 10

<211> 70

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

gctgttgacc tgcggtgatg tagaagaaaa tcctggtcca atggggagaa aagagatgat 60

ggtgagagac 70

<210> 11

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

taacctcgag ttactgcaac caccaccaaa ggctattgag 40

<210> 12

<211> 31

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

ggtggaattc atgcggcccg gcctcccctt c 31

<210> 13

<211> 43

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

ctctagactc gagtcagcat gtcttgagca tgcggtagag cag 43

Claims

1. The application of the cytomegalovirus envelope protein in constructing a lentiviral vector is characterized in that: the cytomegalovirus envelope protein is envelope protein gH, gL and gO, the coding sequence of the envelope protein gH is a nucleotide sequence shown by SEQID NO. 1, the coding sequence of the envelope protein gO is a nucleotide sequence shown by SEQID NO. 3, the gL and the gO are co-expressed, and the nucleotide sequence of the co-expressed gL and the gO is SEQID NO. 2.

2. A lentiviral vector, wherein: the envelope protein in the lentiviral vector is the cytomegalovirus envelope protein in claim 1, the lentiviral vector comprises two plasmids, one is a plasmid for expressing gL and gO, and the other is a plasmid for expressing gH;

the plasmid for expressing gL and gO also comprises a coding sequence of T2A peptide, wherein the coding sequence of the T2A peptide is a nucleotide sequence shown in SEQ ID NO. 4;

the nucleotide sequences of gL and gO are connected through the nucleotide sequence of T2A peptide to form a coexpression structure.

3. Use of the lentiviral vector of claim 2 to prepare a lentiviral pseudovirus.

4. A method for preparing a lentivirus pseudovirus, which is characterized by comprising the following steps: the preparation method comprises the following steps:

(1) Cloning a nucleotide sequence SEQID NO of 2 for co-expressing gL and gO into an expression vector, transfecting cells, and screening packaging cells for stably expressing gL and gO by antibiotics;

(2) Independently cloning a coding sequence SEQID NO 1 of gH into an expression vector, transferring the coding sequence SEQID NO 1 and the coding sequence PLENti, pLP1 and pLP2 into the packaging cells which are obtained by screening in the step (1) and stably express gL and gO, and culturing the cells to generate the lentiviral pseudoviruses with the cytomegalovirus envelope protein;

the expression vector in the step (1) is an MSCV vector; the transfected cells are 293-T cells; the antibiotic is bleomycin;

the expression vector in the step (2) is pcDNA3.1+; the three plasmids of gH, pLenti, pLP1 and pLP2 are transferred into a packaging cell which is obtained by screening and stably expresses gL and gO, the vector expression plasmid is transcribed to generate lentivirus genome mRNA, rev protein is combined with RRE in the lentivirus genome mRNA, the genome mRNA is transferred into cytoplasm from a cell nucleus, and the genome mRNA, capsid protein, reverse transcriptase, integrase and CMV envelope protein are assembled into pseudovirus particles to generate the lentivirus pseudovirus with cytomegalovirus envelope protein.

5. Use of the lentiviral vector of claim 2 and/or the lentiviral pseudovirus produced according to claim 4 for transfecting an NK cell.