Infectious bovine rhinotracheitis delta TK/delta gE gene deletion mark live vaccine and preparation method

A technology for rhinotracheitis and gene deletion, applied in the field of genetic engineering of animal pathogens, can solve the problems that the disease has not attracted enough attention and started late, and achieve the effect of broad market application prospects, real results, and convenient practical operation

Inactive Publication Date: 2012-08-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in our country, due to the late start, the disease has not attracted enough attention, and there is no domestic comme...

Method used

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  • Infectious bovine rhinotracheitis delta TK/delta gE gene deletion mark live vaccine and preparation method
  • Infectious bovine rhinotracheitis delta TK/delta gE gene deletion mark live vaccine and preparation method
  • Infectious bovine rhinotracheitis delta TK/delta gE gene deletion mark live vaccine and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 PCR amplification of EGFP gene expression cassette and TK, gE upstream and downstream homology arms

[0043] According to the full-length nucleotide sequence of the bovine herpesvirus type 1 (BHV-1) genome published on GenBank (http: / / www.ncbi.nlm.nih.gov / ) (Genbank accession number is AJ004801), design 4 pairs Primers (see Table 1 for primer pair sequences, all primers were synthesized by Beijing Aoke Biotechnology Co., Ltd.), using BHV-1 genomic DNA as a template to amplify the TK gene ( figure 2 ) and gE gene ( image 3 ) upstream and downstream homology arms. Plasmid pEGFP-C1 (purchased from Clontec Laboratoties, USA) was double digested with BamH I and Bgl II to remove multiple cloning sites, and T4 ligase was ligated as a template to amplify the EGFP expression cassette. See Table 2 for amplification conditions. A fragment of about 1900bp of the EGFP gene expression cassette was amplified respectively, which was consistent with the expected size (see...

Embodiment 2

[0052] Construction of transfection vector

[0053] The vector plasmid pBluescriptII SK+ (gifted by Zhou Fuchun, School of Veterinary Medicine, Huazhong Agricultural University) was digested with XbaI and EcoR I, respectively, and EcoR I and HindIII (all restriction enzymes were purchased from Bao Biological Engineering (Dalian) Co., Ltd.) cut. The upstream homology arm PCR product of TK was double-digested with XbaI and EcoR I, and the downstream homology arm PCR product was double-digested with EcoR I and Hind III. Ligated to the vector pBluescriptII SK+. Insert the upstream homology arm of TK double-digested with restriction endonuclease XbaI and EcoR I into the upstream cloning vector pBluescriptII SK+, and then insert the downstream homology arm of TK double-digested with EcoR I and Hind III to construct the intermediate transfer Vector, and named pZF08-21 (see figure 1 A). Then insert the EGFP expression cassette to construct a recombinant transfer plasmid, which is ...

Embodiment 3

[0055] Example 3 Construction and Identification of Bovine Infectious Rhinotracheitis ΔTK / ΔgE Gene Deletion Marker Strain

[0056] 1. BHV-1TK - / EGFP + Recombinant virus homologous recombination and screening purification

[0057] Using the calcium phosphate method (Dingming, 2010), the recombinant transfer plasmid pZF07-16 obtained in Example 2, the helper plasmid pBICP0 and the BHV-1 genome were co-transfected into MDBK cells (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) 4h after co-transfection, replace with fresh DMEM medium (preparation method: dissolve commercial 1L DMEM powder in 900mL sterilized ddHO 2 O, add 3.7g NaHCO 3 , adjust the pH value to about 6.8 with HCl, add water to 1000mL, filter and sterilize with a 0.22 μm filter membrane on an ultra-clean bench, and store at 4°C after aliquoting; DMEM powder was purchased from GIBCO BRL Company, item number: 12800 -017). The next day, the cells ...

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Abstract

The invention belongs to the technical field of animal-borne disease genetic engineering and specifically relates to an infectious bovine rhinotracheitis delta TK/delta gE gene deletion mark live vaccine and a preparation method. The strain is preserved at China Center for Type Culture Collection and the preservation number is CCTCCNO: V201104. A recombined infectious bovine rhinotracheitis strain is lack of TK and gE genes and an EGFP (Enhanced Green Fluorescent Protein) express box is inserted. The toxicity of the prepared infectious bovine rhinotracheitis delta TK/delta gE gene deletion mark live vaccine is greatly reduced; the potential virus is difficult to activate; the safety is high; besides, except being lack of the TK and gE genes, the deletion strain can still express the other toxic genes; and the immunogenicity is excellent.

Description

technical field [0001] The invention relates to the technical field of genetic engineering of animal pathogens. Specifically relates to a bovine herpes virus type 1 (BHV-1) thymidine kinase (TK) and gE glycoprotein gene deletion marked live vaccine and a preparation method thereof. Background technique [0002] Infectious Bovine Rhinotracheitis (IBR), commonly known as red nose disease (Li Zuobo, 2006), is an acute, febrile, contact-related disease of cattle caused by bovine herpesvirus type 1 (BHV-1). Infectious diseases mainly cause respiratory and reproductive tract diseases, such as infectious rhinotracheitis, conjunctivitis, high fever, infectious pustular vaginitis, abortion, etc. The occurrence of this disease greatly reduces the milk production of dairy cows, the fecundity of bulls, and the usability of draft cattle, and the respiratory tract infection produced is also prone to secondary bacterial pneumonia in cattle, which is also the cause of economic losses in th...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/265A61P31/20C12R1/93
Inventor 刘正飞定明范强郭爱珍高觉婧陈焕春
Owner HUAZHONG AGRI UNIV
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