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Recombinant lentiviral vector as well as recombinant lentivirus and application thereof

A technology of recombinant lentivirus and lentiviral vector, which is applied in the field of genetic engineering, can solve the problems of no relevant reports, etc., and achieve the effects of improving economic benefits, high activity, and improving fertilization rate

Inactive Publication Date: 2019-03-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As for OPN protein from porcine, no matter whether it is prokaryotic or eukaryotic expression, there is no finished product on the market, and there is no related report at home and abroad.

Method used

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  • Recombinant lentiviral vector as well as recombinant lentivirus and application thereof
  • Recombinant lentiviral vector as well as recombinant lentivirus and application thereof
  • Recombinant lentiviral vector as well as recombinant lentivirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 Construction of recombinant lentiviral vector

[0049] To modify the lentiviral vector pCDH-CMV-MCS-EF1-GFP+Puro, first, synthesize a 160bp double strand containing HSA sequence-enzyme cleavage site-6×His sequence in vitro (shown in SEQ ID NO: 1), Connected to the pMD18-T vector; then, through homologous recombination, design primers HH-F and HH-R to amplify a fragment containing the HSA sequence-restriction site-6×His sequence, with a molecular weight of 160bp ( image 3 A) At the same time, the lentiviral vector was linearized with endonucleases XbaI and BamHI. Finally, the HSA signal peptide (see SEQ ID NO: 5 for its nucleotide sequence) was inserted upstream of the multi-cloning site of the lentiviral vector by seamless connection, and the purification tag 6×His was inserted downstream. Bacterial liquid identification of recombinant cloned plasmids was carried out by lentiviral vector general primers CMV-F and EF1-α-R ( image 3 B), the modified lent...

Embodiment 2

[0052] Embodiment 2, the method for obtaining porcine source OPN recombinant protein

[0053] see figure 1 , is the circuit diagram for obtaining OPN recombinant protein. The first step is the establishment and detection of porcine OPN cell line, and the second step is the acquisition and identification of porcine OPN recombinant protein. The specific method is as follows:

[0054] 1) co-transfect 293FT cells with the lentiviral packaging plasmid pMD2.G, the lentiviral packaging plasmid psPAX2 and the lentiviral recombinant plasmid prepared in Example 1, after transfection for 48h, observe the fluorescence, and then collect the supernatant at 48h and 72h, Centrifuge at 1500rpm for 3min, filter through a 0.45μm filter, concentrate and purify, and obtain a titer of 10 8 TU / mL lentiviral liquid.

[0055] 2) The CHO-K1 cells were revived, and when the cells grew to 80-90%, they were passaged and plated. After 24h, the number of cells grows to approximately 1×10 5 , 6 μg / mL po...

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Abstract

The invention discloses a recombinant lentiviral vector as well as a recombinant lentivirus and application thereof. The recombinant lentiviral vector is prepared by inserting HAS signal peptide intothe upstream of a multiple cloning site of a lentiviral vector, and inserting a purification tag 6*His sequence into the downstream to obtain a converted lentiviral vector; then inserting 6'His-OPN segment between XbaI restriction enzyme cutting site and BamHI restriction enzyme cutting site of the converted lentiviral vector. According to the invention, a CHO cell line is stably converted and purified to obtain an OPN recombinant protein which is conveniently purified; expression time and level can be controlled by people, and the structure of the protein is closer to that of natural protein;the protein has higher activity and more comprehensive functions. By adding the protein into a porcine in vitro fertilization culture line, the fertilization rate can be greatly improved and the development of embryo is improved. The protein can be further applied to boar semen for improving the reproductive rate of porcine. The protein brings a better development prospect for a pig farm and pushes the pig husbandry to a new development stage.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant lentiviral vector, a recombinant lentivirus, Chinese hamster ovary cells containing the recombinant lentivirus and applications thereof. Background technique [0002] Lentivirus is a kind of retrovirus, which has the advantages of large target fragment capacity, high infection rate, and high safety. It can infect quiescent cells and does not produce chimeric animals. Express. Lentiviral vectors have become an effective vehicle for transgenics. Hofmann et al. (2003) used a lentiviral vector with a generalized promoter (LV-PGK) to inject virus particles with the green fluorescent protein gene (GFP) as the target gene into pig embryos. Liu et al. (2013) injected the EGFP gene into the perivitelline space of 46 fertilized eggs with a lentiviral vector, and then combined with embryo transfer technology to give birth to 8 transgenic lambs. EGFP was expressed in all ti...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N7/01C12N5/10C07K14/47
CPCC07K14/47C12N5/0682C12N7/00C12N15/86C12N2510/02C12N2740/15021C12N2740/15043
Inventor 张守全陈云卫恒习赵志宏冯美莹叶超王凯李莉孟立
Owner SOUTH CHINA AGRI UNIV
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