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Recombinant gene of rabies virus, recombinant pseudovirus as well as construction method and application of recombinant pseudovirus

A recombinant gene and rabies virus technology, applied in the field of genetic engineering, can solve the problems of hindering RABV biological research, difficult to obtain pathogens, and high infectivity of rabies virus, and achieves improved immunogenicity, enhanced immunogenicity, and high packaging titer. Effect

Active Publication Date: 2021-07-27
SOUTH CHINA INSTITUDE OF BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the high infectivity of rabies virus, the pathogen is difficult to obtain, thus hampering the biological research on RABV, including antiviral drug research and vaccine development

Method used

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  • Recombinant gene of rabies virus, recombinant pseudovirus as well as construction method and application of recombinant pseudovirus
  • Recombinant gene of rabies virus, recombinant pseudovirus as well as construction method and application of recombinant pseudovirus
  • Recombinant gene of rabies virus, recombinant pseudovirus as well as construction method and application of recombinant pseudovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the design and synthesis of the recombinant gene of rabies virus

[0044] Carry out G-N sequence expression design on the basis of RABV-G gene sequence (KT221113.1), add Nhe I and Mlu I enzyme cutting sites before the start codon of the expression frame and after the stop codon, obtain such as SEQ ID NO : Nucleotide sequence shown in 1.

[0045] According to the nucleotide sequence shown in SEQ ID NO:1, Anhui General Biotechnology Co., Ltd. was entrusted to use a gene synthesizer to artificially synthesize the recombinant gene of the rabies virus of the present invention. The amino acid sequence of the recombinant gene is shown in SEQ ID NO:2.

Embodiment 2

[0046] Embodiment 2, construct recombinant plasmid and package recombinant pseudovirus

[0047] 1. Construction of recombinant plasmids:

[0048] S1. Insert the recombinant gene of the rabies virus prepared in Example 1 into the pUC57 vector to form vector I, and name the vector I as pUC-RABV-G-N;

[0049] S2. Recover small fragments after pUC-RABV-G-N was digested with Nhe I and Mlu I; the lentiviral envelope plasmid pCMV-VSV-G (from Thermo Fisher Scientific (China) Co., Ltd.) was subjected to Xho I single Enzyme digestion and 2% agarose gel electrophoresis, gel recovery of large fragments;

[0050] S3. Fill in the large fragment recovered by enzymatic digestion of the lentiviral envelope plasmid pCMV-VSV-G and obtain fragment I by dephosphorylation, and fill in the small fragment recovered by enzymatic digestion of pUC-RABV-G-N to obtain fragment II, and then use The T4 ligase of NEB connects Fragment I and Fragment II to construct a recombinant envelope plasmid named pC...

Embodiment 3

[0057] Embodiment 3, a kind of composition

[0058] A composition comprising the recombinant pseudovirus prepared in Example 2 above and an adjuvant, the adjuvant may include CpG-ODN.

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Abstract

The invention relates to the technical field of gene engineering, in particular to a recombinant gene of a rabies virus, a recombinant pseudovirus and a construction method and application of the recombinant pseudovirus. The nucleotide sequence of the recombinant gene is as shown in SEQ ID NO: 1. The preparation method comprises the following steps: replacing VSV-G in a lentivirus envelope plasmid with a recombinant gene RABV-G-N to construct a recombinant envelope plasmid pCMV-RABV-G-N, and then co-transfecting a human embryonic kidney cell HEK-293T with the recombinant envelope plasmid, pLV-eGFP containing a green fluorescent protein reporter gene and psPAX2 to obtain the recombinant pseudovirus. The recombinant gene provided by the invention has biological characteristics of natural rabies virus, and can improve pseudovirus immunogenicity, and the prepared recombinant pseudovirus has high packaging titer, and can replace natural rabies virus to evaluate serum neutralizing antibody titer.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant gene of rabies virus, a recombinant pseudovirus and a construction method and application thereof. Background technique [0002] Rabies is a contagious zoonotic disease caused by rabies virus (RABV) invading the central nervous system, and is mainly transmitted by infected animals through saliva and bites. Rabies remains a serious public health problem in most countries in Asia and Africa. [0003] Rabies is mainly prevented and intervened by vaccination of rabies vaccine. At present, the vaccines produced in China mainly come from inactivated rabies virus vaccines cultured in Vero cells, followed by inactivated virus vaccines derived from human diploid cells and hamster kidney cells. Other types of rabies vaccines are approved for use. The production of inactivated rabies virus vaccine in cells needs to continuously replicate the virus to achieve high...

Claims

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Application Information

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IPC IPC(8): C12N15/47C12N15/867C12N15/66C12N5/10C12N7/01A61K39/205A61K39/39A61P31/14C12R1/93
CPCC07K14/005C12N15/86C12N15/66C12N7/00A61K39/12A61K39/39A61P31/14C12N2740/15043C12N2760/20122C12N2760/20152C12N2740/15022C12N2740/15052A61K2039/55561C12N2760/20134
Inventor 帅丽芳许明宇李红卫何跃忠武旺盛何敏杰毛莹莹颜仁和梁文翰陈泽典
Owner SOUTH CHINA INSTITUDE OF BIOMEDICINE
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