Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus

A technology of bovine parainfluenza virus, foot-and-mouth disease virus, applied in the fields of molecular biology and virology

Inactive Publication Date: 2014-05-07
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral promoters are present at the 3' end of the genomic RNA, and the GS and GE restriction regions of each gene usually consist of short sequences of no more than 200 nucleotides (De et al., 1990), so the cis-acting signal is relatively short

Method used

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  • Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus
  • Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus
  • Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus

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Experimental program
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Effect test

Embodiment 1

[0028] 1.2 Construction of recombinant BPIV3 full-length cDNA clone

[0029] The virus liquid that BPIV3 virus inoculates MDBK cell harvest extracts virus genome RNA by routine method (Trizol method); figure 1 The entire genome shown is divided into 7 partially overlapping fragments (S1, S2.1, S2.2, S3.1, S3.2, S4.1, S4.2) according to the restriction site for RT-PCR amplification , the PCR method was introduced at the 5' end of S1 Nhe I restriction endonuclease site and HamRz ribozyme sequence, the 3' end of S4.2 introduces HdvRz ribozyme sequence and not I restriction endonuclease site. The amplified fragment was cloned into the pCR-Blunt vector and sequenced to confirm that the sequence was consistent with the viral RNA sequence. The complete genome cDNA of BPIV3 was assembled by ligation of the restriction sites of overlapping parts of adjacent fragments, and cloned in the transcription vector pcDNA3.1 Nhe I and not Between I, the full-length cDNA clone pcDNA-N...

Embodiment 2

[0050] 1 1. Materials and methods

[0051] 1.1 Materials

[0052]NM09 strain of bovine parainfluenza virus type 3 was isolated, identified and preserved by the Institute of Specialty Products, Chinese Academy of Agricultural Sciences; Preservation; MDBK, BSR cells were cultured in monolayer in DMEM (Gibco BRL) medium containing 10% FBS (Gibco BRL); viruses were propagated in DMEM maintenance solution with 2% FBS; Trizol Reagent, Pfx DNAPolymerase, Zero Blunt PCR Cloning Kit, plasmid pcDNA3.1 (+) and transfection reagent LipofectamineTM2000 Reagent were purchased from Invirtrogen; T4 DNA ligase and restriction endonuclease were purchased from NEB; TaKaRa Taq DNA polymerase, dNTP Mixture, random primers, and DL15000 Marker were purchased from Bought from Dalian Bao Biotechnology Engineering Co., Ltd.; M-MLV reverse transcriptase and RNase inhibitor were purchased from Promega; gel recovery kit and plasmid extraction kit were purchased from Aisigen Biotechnology (Hangzhou) Co.,...

Embodiment 3

[0053] 1.2 Construction of the full-length cDNA clone of the recombinant bovine parainfluenza virus genome expressing the gene of porcine O-type foot-and-mouth disease virus VP1 protein.

[0054] Using the cloning of the above-mentioned BPIV3 full-length segmented S2 fragment as an intermediate vector, carry out base point-directed mutation at 3635-3640nt of the full-length cDNA of the BPIV3 genome by PCR method, and introduce Age I restriction endonuclease sites, using pas I digestion point, and then re-insert the mutated S2 fragment back into the pcDNA-NM09 full-length cDNA clone to obtain the BPIV3 recombinant transcription vector pcDNA-NM09-AgeI (P / M); -PCR is introduced respectively at the 5' end and 3' end of the coding region of the foot-and-mouth disease virus VP1 gene Age I restriction endonuclease recognition site, initiation codon and termination codon, and the transcription termination sequence (AATTAAGAAAAA) and transcription initiation sequence ( AGGATAAAAG...

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Abstract

The invention relates to a recombined cattle parainfluenza carrier pcDNA-NM09-VP1 for expressing the protein VP1 of a porcine O type foot-and-mouth disease virus. The recombined cattle parainfluenza carrier is characterized in that an RNA (ribonucleic acid) extracted from a strain BPIV3NM09 is used as a template; a virus total-length gene group is segmentally amplified through RT-PCR (reverse transcription-polymerase chain reaction); the total-length cDNA of cattle parainfluenza is subjected to primary modification, namely AgeI is introduced between P and M, so that insertion and replacement of exogenous antigen gene fragments are facilitated; due to secondary modification, antigen epitope which is inserted into a heterologous virus in a modified manner is the protein VP1 of the O type foot-and-mouth disease virus; the amino acid sequence is SEQIDNO1, and the nucleotide sequence is SEQIDNO2. The foundation is laid for further development of a gene engineering recombined vaccine for preventing and controlling the porcine foot-and-mouth disease and research on the III-type toxicity factor and the molecular pathogenesis of the cattle parainfluenza; the recombined cattle parainfluenza carrier has the replication capacity.

Description

technical field [0001] The invention relates to a recombinant bovine parainfluenza virus vector expressing porcine foot-and-mouth disease virus type O VP1 protein, in particular to a method for constructing a recombinant bovine parainfluenza virus type 3 vector expressing porcine foot-and-mouth disease virus structural protein VP1 gene and a biological method of the recombinant virus It belongs to the field of molecular biology and virology. Background technique [0002] The virulence of parainfluenza virus is weakened due to the limitation of natural host range for different kinds of animals. Therefore, the protective antigen gene of the heterologous virus can be inserted into the attenuated parainfluenza virus as a vector to express the protective antigen of the heterologous virus, and the protective antigen of the human respiratory syncytial virus can be expressed by using BPIV3 as the vector , which can maintain the weak toxicity of BPIV3 in the human body and maintai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86
Inventor 王凤雪武华温永俊师新川张淑琴王炜
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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