Recombinant chimpanzee source adenovirus for expressing rabies virus G protein and preparation method of recombinant chimpanzee source adenovirus

A rabies virus and chimpanzee technology, applied in the field of recombinant chimpanzee-derived adenovirus and its preparation, can solve problems such as no reported rabies virus vaccine, achieve excellent immune protection effect, efficiently induce immune response, and produce safe and convenient effects.

Pending Publication Date: 2022-06-07
长睿生物技术(成都)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there is no report of a rabies virus vaccine expressing rabies virus G protein developed by chimpanzee adenovirus

Method used

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  • Recombinant chimpanzee source adenovirus for expressing rabies virus G protein and preparation method of recombinant chimpanzee source adenovirus
  • Recombinant chimpanzee source adenovirus for expressing rabies virus G protein and preparation method of recombinant chimpanzee source adenovirus
  • Recombinant chimpanzee source adenovirus for expressing rabies virus G protein and preparation method of recombinant chimpanzee source adenovirus

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Experimental program
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Effect test

Embodiment 1

[0066] This embodiment provides a method for constructing an E1-deleted replication-deficient adenoviral vector pAdC68 (pAdC68-E1-deleted), which specifically includes the following steps:

[0067] (1) Construction of plasmid pNEB193-KE

[0068] According to the multiple cloning site on the pNEB193 vector and the sequence of the adenovirus AdC68 genome, the primers for amplifying the KE fragment on the adenovirus AdC68 genome (refer to Table 1) were designed, and the target product KE fragment was amplified by PCR. The total volume of the PCR amplification system was 50 μL, and the reaction cycle parameters were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 1 min; annealing temperature at 57°C for 30 s; extension at 72°C for 2.2 min. The KE fragment amplified by EcoRI and KpnI double enzyme digestion PCR, the KE fragment was purified by agarose gel, connected to the pNEB193 vector that had been digested by the same enzyme, transformed into DH5α competent cells,...

Embodiment 2

[0085] In this embodiment, the G protein-encoding gene Gp gene of rabies virus and the WPRE regulatory element are linked into pAdC68-E1-deleted in Example 1.

[0086] (1) According to the nucleic acid sequence of the Gp coding region in the rabies virus ERA strain in GenBank, the codon-optimized Gp gene (as shown in SEQ ID NO.1) was synthesized, and the WPRE sequence was inserted at the 3', and the optimized The 5' end of the target gene of the codon is inserted into EcoRI and NheI restriction sites in turn, and the 3' end is connected to the XbaI restriction site ( figure 1 ), the optimized Gp gene was cloned into the commercially available vector pUC57 vector by double digestion with EcoRI and XbaI to obtain the recombinant plasmid pUC57-0 / Gp-WPRE.

[0087] (2) The pUC57-0 / Gp-WPRE plasmid was digested with NheI and XbaI to form the target gene with cohesive ends, and the adenovirus shuttle vector pShuttle-CMV was digested with NheI and XbaI to obtain a linearized vector , ...

Embodiment 3

[0090] The recombinant adenoviral plasmid pAdC68-Gp-WPRE in Example 2 was linearized with the restriction endonuclease PacI, and the plasmid was transferred into HEK293 cells (6-well plate) by lipofection, and incubated at 37°C for 6- On day 8, obvious plaques appeared. After the cells became round and suspended, the cells were collected, and after repeated freezing and thawing three times, the virus supernatant was taken to infect HEK293 cells (75ml cell culture flask). Repeat the above steps until an appropriate amount of virus is collected (about 20-40 150ml cell culture flasks), purify the virus by cesium chloride density gradient centrifugation, measure the OD value, add glycerol with a final concentration of 10% and store at -80°C. Extract viral genomic DNA (virus content 10 12 vp), and BglII, BamHI, NheI restriction map analysis. Use the CMV promoter universal primer to detect the nucleic acid sequence of the target fragment. The purified virus was passed continuousl...

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Abstract

The invention discloses a recombinant chimpanzee adenovirus for expressing rabies virus G protein and a preparation method of the recombinant chimpanzee adenovirus, and relates to the technical field of biology. The adenovirus comprises a Gp gene after codon optimization, a WPRE after transcription regulation element and an AdC68 genome sequence containing KE, AK, XA and PX fragments, and a connecting sequence is inserted between the 594th site and the 3513th site in the AdC68 genome to replace a part of E1 sequence between the 459th site and the 3011th site. The codon of the Gp gene is optimized, and a WPRE transcriptional regulatory element is inserted, so that the high-level secretory expression of the rabies virus G protein is realized. The structure and function of the obtained rabies virus G protein are completely consistent with those of a natural state, the immunogenicity of the G protein is reserved to the maximum extent, and the G protein can be better recognized by antigen presenting cells. And the prepared rabies vaccine also has high immunogenicity, low prestored antibody property and safety.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant chimpanzee-derived adenovirus expressing rabies virus G protein and a preparation method thereof. Background technique [0002] Rabies is a serious comorbid but preventable infectious disease of humans and animals that can cause severe encephalitis in warm-blooded animals. Infected persons who have not received vaccine immunization will almost certainly die when neurological symptoms appear. The usual cause of death is that the central nervous system (brain-spinal cord) is destroyed by the virus. A large number of viruses exist in the cerebrospinal fluid, saliva and body fluids of patients, and most of them are transmitted through bites. According to statistics, rabies causes 26,000-55,000 deaths worldwide every year, of which more than 95% of the deaths occur in Asia and Africa, and the vast majority (97%) of the deaths are caused by dogs. [0003] The rabies virus e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/47C12N15/861A61K39/205A61P31/14
CPCC12N7/00C07K14/005C12N15/86A61K39/12A61P31/14C12N2800/22C12N2840/60C12N2710/10321C12N2710/10343C12N2710/10334C12N2710/10352C12N2760/20122A61K2039/5256A61K2039/575C12N2760/20134Y02A50/30
Inventor 杨军邓飞郑飞刘璐
Owner 长睿生物技术(成都)有限公司
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