阅读说明：本技术 一种重组慢病毒载体、重组慢病毒 (recombinant lentivirus vectors and recombinant lentiviruses ) 是由 王磊 张德庆 胡俊 杨国华 于 2019-06-20 设计创作，主要内容包括：本发明涉及基因工程技术领域,尤其是一种重组慢病毒载体、重组慢病毒,重组慢病毒载体主要以慢病毒载体pCDH-CMV-MCS-EF1-GFP+Puro为骨架,在慢病毒载体pCDH-CMV-MCS-EF1-GFP+Puro上插入信号肽序列S,并通过重组慢病毒载体将293T细胞转染成慢病毒后感染细胞,并转染成本远远低于脂质体,感染效率高；另外,可以筛选出过表达稳定细胞株,进行无血清悬浮培养,分泌表达目的蛋白,能大大提高蛋白产量,有效降低杂蛋白比例；另外,通过引入His标签,从而有利于表达蛋白的检测和纯化。(The invention relates to the technical field of genetic engineering, in particular to recombinant lentiviral vectors and recombinant lentiviruses, wherein the recombinant lentiviral vectors mainly take lentiviral vectors pCDH-CMV-MCS-EF1-GFP + Puro as a framework, a signal peptide sequence S is inserted into the lentiviral vectors pCDH-CMV-MCS-EF1-GFP + Puro, 293T cells are transfected into lentiviruses through the recombinant lentiviral vectors and then infected into the cells, the transfection cost is far lower than that of liposome, the infection efficiency is high, in addition, overexpression stable cell strains can be screened out for serum-free suspension culture, the target protein is secreted and expressed, the protein yield can be greatly improved, the impurity protein proportion is effectively reduced, and in addition, the detection and purification of the expressed protein are facilitated by introducing a His label.)

The recombinant lentivirus vectors and recombinant lentiviruses are characterized in that the recombinant lentivirus vectors mainly use lentivirus vectors pCDH-CMV-MCS-EF1-GFP + Puro as frameworks, and signal peptide sequences S are inserted into the lentivirus vectors pCDH-CMV-MCS-EF1-GFP + Puro, and the specific implementation process is as follows;

step 1: carrying out XbaI and EcoRI double enzyme digestion on the vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, recovering a vector framework part, and designing and synthesizing a signal peptide sequence S with XbaI and EcoRI cohesive ends;

step 2: connecting the carrier skeleton part with a signal peptide sequence S by using ligase to obtain a recombinant carrier, namely pCDH-S;

and step 3: designing an upstream primer at a NotI enzyme cutting site of a recombinant vector pCDH-S, and inserting a purification tag 6 × His sequence and a stop codon to obtain a modified lentiviral vector pCDH-CMV-HSA-MCS-6His-EF1-GFP + Puro;

and 4, step 4: inserting the 6His-OPN fragment into the position between the XbaI and EcoRI double enzyme cutting sites of the modified lentivirus vector, designing a downstream primer at the PstI enzyme cutting site, taking the recombinant vector pCDH-S as a template, and obtaining a PCR product containing a His label through PCR reaction;

and 5: the PCR product containing the His tag and the recombinant vector pCDH-S are respectively subjected to NotI and PstI double enzyme digestion and then connected to construct a recombinant lentiviral vector pCDH-CMV-HSA-6' His-OPN-6His-EF1-GFP + Puro.

2. The recombinant lentiviral vector and recombinant lentivirus of claim 1, wherein the signal peptide sequence S is formed by annealing two complementary oligonucleotide strands with XbaI and EcoRI cleavage sites, and the amino acid sequence of the S signal peptide is UCGCAGAUCCUUCGCGG.

3. The recombinant lentiviral vector and recombinant lentivirus of claim 1, wherein in the step 2, the ligase is ligated to the signal peptide sequence S, wherein the amino acid sequence of the peptide chain of the ligase replicates the amino acid sequence of the signal peptide sequence S, and the replicated amino acid sequence is AGCGTCTAGGAAGCGGC and is inserted into the peptide chain of the ligase by hydrogen bonding.

4. The recombinant lentiviral vector and recombinant lentivirus of claim 1, wherein in step 3, the sequence of the purification tag 6 XHis is GTTGATCTCAGAAGACGCACTCTC and the stop codon is UAG.

5. The recombinant lentiviral vector and recombinant lentivirus of claim 1, wherein in step 5, the NotI and PstI double-enzyme cut amino acid sequences are CGCAAATGGGCGTAGGCGTG and GGACTGTGGGCGATGTGC, respectively, and the amino acid sequences after the disconnection are recombined with the amino acid sequence of the His-tag-containing PCR product to finally recombine into pCDH-CMV-HSA-6' His-OPN-6His-EF1-GFP + Puro.

The use of the recombinant lentiviral vector and the recombinant lentivirus of claims 1 to 5, wherein the recombinant lentiviral vector can infect 293T cells after transforming into lentivirus, and the specific transfection process is as follows:

(1) greatly extracting the recombinant plasmid pCDH-S-PLGF-His, the two auxiliary plasmids pSPAX2 and pMD2.G to obtain a high-concentration high-purity endotoxin-free plasmid;

(2) culturing 293T cells by a conventional method, stably passaging the cells, and then inoculating the cells to a 60mm culture dish, wherein the number of the cells is based on 85% confluency after 24 hours of culture;

(3) plasmids pCDH-S-PLGF-His, pSPAX2 and pMD2.G were made according to 4: 3: 1 proportion of 8 mu g of plasmid is added into 500 mu of LPBS, the mixture is mixed gently, 20 mu of PEI is added into 500 mu of PBS, the mixture is mixed by vortex, the PEI mixed solution is added into the plasmid, the mixture is mixed gently and placed for 20min at room temperature;

(4) sucking 1mL of 293T cell culture medium, adding the mixed solution into the cells, culturing overnight or changing the solution after 12h, continuously culturing for 48h, then harvesting the supernatant, and simultaneously supplementing fresh culture medium and continuously culturing for 24h, and then harvesting the supernatant. During the process, the green fluorescence condition of the cells is observed under a fluorescence microscope, so that the transfection process is normal, and the transfection efficiency is normal.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to recombinant lentiviral vectors and recombinant lentiviruses.

Background

Lentivirus refers to kinds of virus vectors from human immunodeficiency virus-1 (HIV-1), the lentivirus vector contains genetic information required by packaging, transfection and stable integration and is a main component of a lentivirus vector system, the lentivirus vector system mainly comprises three different plasmids, namely transfer plasmids, auxiliary plasmids and envelope expression plasmids, the transfer plasmids are mainly used for inserting exogenous target genes and simultaneously retain elements which play a main role in the integrated replication of the virus, the auxiliary plasmids mainly comprise gag, pol and rev proteins, the gag is mainly a core protein for encoding the virus, the pol is mainly used for encoding enzymes required in the viral replication process, the rev protein is used for participating in a nuclear-plasma transport process for encoding mRNA of HIV structural protein, the signal peptide refers to 15-30 amino acids at the N-terminal end of a secretory protein precursor and has a specific role in polypeptide chain synthesis, the function of the signal peptide is mainly used for attaching translated ribosome to a RER membrane, the protein is guided in cells, the protein is used for intracellular transport, the signal peptide is extracted from a large amount by a conventional cell transfection system, the signal peptide is extracted, and the purity of the signal peptide is required for cell culture, and the purity is low.

Disclosure of Invention

The invention aims to solve the defect that the prior art has a plurality of hybrid proteins of extracted protein-lysed cells, and provides recombinant lentiviral vectors and recombinant lentiviruses.

In order to achieve the purpose, the invention adopts the following technical scheme:

recombinant lentiviral vectors and recombinant lentiviruses are designed, the recombinant lentiviral vectors mainly take lentiviral vectors pCDH-CMV-MCS-EF1-GFP + Puro as a framework, and a signal peptide sequence S is inserted into the lentiviral vectors pCDH-CMV-MCS-EF1-GFP + Puro, and the specific implementation process is as follows;

step 1: carrying out XbaI and EcoRI double enzyme digestion on the vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, recovering a vector framework part, and designing and synthesizing a signal peptide sequence S with XbaI and EcoRI cohesive ends;

step 2: connecting the carrier skeleton part with a signal peptide sequence S by using ligase to obtain a recombinant carrier, namely pCDH-S;

and step 3: designing an upstream primer at a NotI enzyme cutting site of a recombinant vector pCDH-S, and inserting a purification tag 6 × His sequence and a stop codon to obtain a modified lentiviral vector pCDH-CMV-HSA-MCS-6His-EF1-GFP + Puro;

and 4, step 4: inserting the 6His-OPN fragment into the position between the XbaI and EcoRI double enzyme cutting sites of the modified lentivirus vector, designing a downstream primer at the PstI enzyme cutting site, taking the recombinant vector pCDH-S as a template, and obtaining a PCR product containing a His label through PCR reaction;

and 5: the PCR product containing the His tag and the recombinant vector pCDH-S are respectively subjected to double enzyme digestion through NotI and PstI and then are connected to construct a recombinant lentiviral vector pCDH-CMV-HSA-6' His-OPN-6His-EF1-GFP + Puro;

preferably, the signal peptide sequence S is formed by annealing two complementary oligonucleotide strands with XbaI and EcoRI cleavage sites, and the amino acid sequence of the S signal peptide is UCGCAGAUCCUUCGCGG.

Preferably, in step 2, the ligase is ligated to the signal peptide sequence S, the amino acid sequence of the peptide chain of the ligase replicates the amino acid sequence of the signal peptide sequence S, and the replicated amino acid sequence is AGCGTCTAGGAAGCGGC and is inserted into the peptide chain of the ligase by hydrogen bonding.

Preferably, in step 3, the sequence of the purification tag 6 × His is GTTGATCTCAGAAGACGCACTCTC, and the stop codon is UAG.

Preferably, in step 5, the NotI and PstI double-enzyme cut amino acid sequences are CGCAAATGGGCGTAGGCGTG and GGACTGTGGGCGATGTGC, respectively, and the amino acid sequences in the cut-off are recombined with the sequence of the amino acid of the His-tag-containing PCR product to finally recombine into pCDH-CMV-HSA-6' His-OPN-6His-EF1-GFP + Puro.

The invention also provides recombinant lentivirus vectors and application of the recombinant lentiviruses according to claims 1-5, wherein 293T cells can be infected after being transfected into lentiviruses by the recombinant lentivirus vectors, and the specific transfection process is as follows:

(1) greatly extracting the recombinant plasmid pCDH-S-PLGF-His, the two auxiliary plasmids pSPAX2 and pMD2.G to obtain a high-concentration high-purity endotoxin-free plasmid;

(2) culturing 293T cells by a conventional method, stably passaging the cells, and then inoculating the cells to a 60mm culture dish, wherein the number of the cells is based on 85% confluency after 24 hours of culture;

(3) plasmids pCDH-S-PLGF-His, pSPAX2 and pMD2.G were made according to 4: 3: 1 proportion of 8 mu g of plasmid is added into 500 mu L of PBS, the mixture is mixed gently, 20 mu g of PEI is added into 500 mu L of PBS, the mixture is mixed by vortex, the PEI mixed solution is added into the plasmid, the mixture is mixed gently and placed for 20min at room temperature;

(4) sucking 1mL of 293T cell culture medium, adding the mixed solution into the cells, culturing overnight or changing the solution after 12h, continuously culturing for 48h, then harvesting the supernatant, and simultaneously supplementing fresh culture medium and continuously culturing for 24h, and then harvesting the supernatant. During the process, the green fluorescence condition of the cells is observed under a fluorescence microscope, so that the transfection process is normal, and the transfection efficiency is normal.

The recombinant lentivirus vectors and recombinant lentiviruses provided by the invention have the beneficial effects that:

1. according to the invention, the cell is infected after the lentiviral vector and the auxiliary plasmid with the signal peptide are packaged into the lentivirus by transfecting 293T cell with PEI, the transfection cost is far lower than that of liposome, and the infection efficiency is high; in addition, an over-expression stable cell strain can be screened out, serum-free suspension culture is carried out, and a target protein is secreted and expressed, so that the protein yield can be greatly improved, and the proportion of hybrid proteins is effectively reduced;

2. the invention is beneficial to the detection and purification of the expression protein by introducing the His label, and effectively solves the problems that the prior lentiviral vector has no label and the expression protein is not beneficial to purification.

Drawings

FIG. 1 is a base sequence chart of recombinant lentivirus vectors and recombinant lentiviruses provided by the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments, not all embodiments, of the present invention .

Referring to FIG. 1, recombinant lentiviral vectors and recombinant lentiviruses, wherein the recombinant lentiviral vectors mainly use a lentiviral vector pCDH-CMV-MCS-EF1-GFP + Puro as a framework, and a signal peptide sequence S is inserted into the lentiviral vector pCDH-CMV-MCS-EF1-GFP + Puro, and the specific implementation process is as follows;

step 1: carrying out XbaI and EcoRI double enzyme digestion on a vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro, recovering a vector framework part, designing and synthesizing a signal peptide sequence S with XbaI and EcoRI cohesive ends, wherein the signal peptide sequence S is formed by annealing two complementary oligonucleotide chains with XbaI and EcoRI enzyme digestion sites, and the amino acid sequence of the S signal peptide is UCGCAGAUCCUUCGCGG;

step 2: connecting a carrier skeleton part with a signal peptide sequence S by using ligase to obtain a recombinant carrier, namely pCDH-S, wherein in the process of connecting the ligase with the signal peptide sequence S, the amino acid sequence of the signal peptide sequence S can be copied on the amino acid sequence of a peptide chain of the ligase, and the copied amino acid sequence is AGCGTCTAGGAAGCGGC and is inserted into the peptide chain of the ligase through a hydrogen bond;

and step 3: designing an upstream primer at a NotI enzyme cutting site of a recombinant vector pCDH-S, and inserting a purification tag 6 XHis sequence and a stop codon to obtain a modified lentiviral vector pCDH-CMV-HSA-MCS-6His-EF1-GFP + Puro, wherein the sequence of the purification tag 6 XHis GTTGATCTCAGAAGACGCACTCTC, and the stop codon is UAG;

and 4, step 4: inserting the 6His-OPN fragment into the position between the XbaI and EcoRI double enzyme cutting sites of the modified lentivirus vector, designing a downstream primer at the PstI enzyme cutting site, taking the recombinant vector pCDH-S as a template, and obtaining a PCR product containing a His label through PCR reaction;

and 5: the PCR product containing the His tag and the recombinant vector pCDH-S are respectively subjected to double enzyme digestion through NotI and PstI and then are connected to construct a recombinant lentiviral vector pCDH-CMV-HSA-6' His-OPN-6His-EF1-GFP + Puro; the NotI and PstI double-enzyme-cleaved amino acid sequences were CGCAAATGGGCGTAGGCGTG and GGACTGTGGGCGATGTGC, respectively, and the amino acid sequences in the cleavage were recombined with the amino acid sequence of the His-tag-containing PCR product to finally recombine into pCDH-CMV-HSA-6' His-OPN-6His-EF1-GFP + Puro.

The invention also provides recombinant lentivirus vectors and application of the recombinant lentiviruses according to claims 1-5, which are characterized in that 293T cells can be infected after being transfected into lentiviruses by the recombinant lentivirus vectors, and the specific transfection process is as follows:

(1) greatly extracting the recombinant plasmid pCDH-S-PLGF-His, the two auxiliary plasmids pSPAX2 and pMD2.G to obtain a high-concentration high-purity endotoxin-free plasmid;

(2) culturing 293T cells by a conventional method, stably passaging the cells, and then inoculating the cells to a 60mm culture dish, wherein the number of the cells is based on 85% confluency after 24 hours of culture;

(3) plasmids pCDH-S-PLGF-His, pSPAX2 and pMD2.G were made according to 4: 3: 1 proportion of 8 mu g of plasmid is added into 500 mu L of PBS, the mixture is mixed gently, 20 mu g of PEI is added into 500 mu L of PBS, the mixture is mixed by vortex, the PEI mixed solution is added into the plasmid, the mixture is mixed gently and placed for 20min at room temperature;

(4) sucking 1mL of 293T cell culture medium, adding the mixed solution into the cells, culturing overnight or changing the solution after 12h, continuously culturing for 48h, then harvesting the supernatant, and simultaneously supplementing fresh culture medium and continuously culturing for 24h, and then harvesting the supernatant. During the process, the green fluorescence condition of the cells is observed under a fluorescence microscope, so that the transfection process is normal, and the transfection efficiency is normal.

In the process of transfecting 293T cells into lentivirus infected cells, constructing a pCDH-S-PEDF-His recombinant plasmid, mainly taking a recombinant lentivirus vector pCDH-S-His as a framework, carrying out double enzyme digestion by EcoRI and NotI to recover the framework part, designing a primer to amplify PEDF genes, wherein the sequence of an upstream primer is 5 ' -TTTGAATTCTATGCAGGCCCTGGTGCTA-3, and the sequence of a downstream primer is 5'-TTTGCGGCCGCGGGGCCCCTGGGGTCCAGAA-3' (SEQ ID NO. 8); the specific implementation steps are as follows:

firstly, taking human cDNA as a template, and obtaining PEDF gene through PCR reaction;

then, the amplification procedure is pre-denaturation at 95 ℃ for 4 minutes, and then denaturation at 95 ℃ for 30 seconds;

annealing at 55 ℃ for 30 seconds, continuing to extend at 72 ℃ for 1 minute and 30 seconds, and circulating for 35 times, extending at 72 ℃ for 10 minutes;

and finally, purifying the PCR product, performing double enzyme digestion by EcoRI and NotI, running gel and recovering, connecting the PCR product with the backbone part of the pCDH-S-His vector recovered by enzyme digestion by using ligase, transforming escherichia coli, selecting a monoclonal colony, culturing, extracting plasmid, and performing sequencing verification. Finally, the recombinant plasmid pCDH-S-PEDF-His is obtained.

According to the invention, the cell is infected after the lentiviral vector and the auxiliary plasmid with the signal peptide are packaged into the lentivirus by transfecting 293T cell with PEI, the transfection cost is far lower than that of liposome, and the infection efficiency is high; in addition, an over-expression stable cell strain can be screened out, serum-free suspension culture is carried out, and a target protein is secreted and expressed, so that the protein yield can be greatly improved, and the proportion of hybrid proteins is effectively reduced; in addition, the introduction of a His tag facilitates the detection and purification of the expressed protein.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.