Construction and application of porcine respiratory and reproductive syndrome virus replication-defective virus vaccine strain
A virus vaccine and virus replication technology, applied in the direction of antiviral agents, viruses/phages, virus antigen components, etc., can solve the problems of poor immune efficacy, strong virulence of live vaccines, economic losses, etc., and achieve good genetic stability. Effect
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Embodiment 1
[0031] Example 1: Construction of recombinant vector pACYC-Jiangxi-3d102 containing Nsp9 deletion coding region
[0032] In this example, the full-length cDNA of PRRSV Jiangxi-3 genome deleted a nucleotide fragment with a length of 102 bp in the Nsp9 coding region, and cloned it into the plasmid pACYC177 vector to obtain a recombinant vector. The nucleotide sequence of the nucleotide fragment is shown at positions 8241 to 8342 from the 5' end of GenBank Accession Number EU200961.
[0033] 1. Construction of the recombinant vector containing the nucleotide fragment of the Nsp9 deletion coding region
[0034] According to the complete sequence of Jiangxi-3 (GenBank sequence number EU200961) and the restriction site of plasmid vector pACYC177, the cloning and detection primers (as shown in Table 1) were designed by software, and the primers were synthesized by Shanghai Yingjun Biotechnology Company.
[0035] Table 1 is used to construct the primer sequence of PRRSV Jiangxi-3 str...
Embodiment 2
[0053] Embodiment 2: the construction of Nsp9 stable expression cell line
[0054] 1. Construction of recombinant plasmid pIRESpuro-Nsp9
[0055] The eukaryotic expression plasmid pIRESpuro2 was purchased from Clonetec Company of the United States; Marc-145 cells were purchased from ATCC Company of the United States. Design a pair of primers according to the Jiangxi-3 (EF398047) Nsp9 gene sequence in GenBank: Nsp9-F: 5′-ATC GCTAGC ATGCTAGCCGCCAGCGGCT, Nsp9-R: 5′-ATC GCGGCCGCTTATTCTCATGATTGGACCTGAGT. In order to facilitate cloning, NheI and NotI restriction enzyme sites were introduced into the primers Nsp9-F and Nsp9-R respectively (underlined are the restriction enzyme sites), and the primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd. PCR amplification of Nsp9 gene, NheI and NotI restriction enzyme digestion ligation of Nsp9 gene fragment and plasmid pIRESpuro2 were carried out according to conventional methods, and Escherichia coli DH5α competent cells...
Embodiment 3
[0061] Embodiment 3: the preparation of replication defective virus
[0062] 1. Transfection and rescue of replication defective virus
[0063] Marc-145-Nsp9 cells were subcultured in 6-well plates, cultured overnight with RPMI-1640 medium containing 10% calf serum by volume, and used for transfection when they reached 60%-70% confluence and uniformity. The plasmid pACYC-CMV-Jiangxi-3d102 used for transfection was extracted with the Spin MiniPrep Kit kit from QIAGEN, USA, and then the content of the plasmid was measured with a spectrophotometer. The plasmid extraction steps were performed according to the instructions of the plasmid extraction kit. The transfection method was according to Lipofectamine from Invitrogen Company of the United States TM 2000 transfection kit operating instructions. 24 hours after transfection, take the transfection supernatant and inoculate a monolayer of Marc-145-Nsp9 cells. After inoculation, add RPMI-1640 medium containing 2% serum by volume...
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