SARS vaccine based on replicative vaccinia virus vector
A technology of vaccinia virus and replication type, which is applied in the field of vaccine against SARS-CoV and its preparation, can solve the problems of high requirements and biological safety hazards
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Embodiment 1
[0047] Example 1: Construction of vaccinia virus universal transfer vector pVTT 1.0
[0048] 1. Construction of recombinant plasmid pSC-neo
[0049] Plasmid pIRESneo (purchased from Clontech Company) was first digested with XhoI, then digested with SmaI (reaction temperature: 25°C), filled in with Klenow enzyme, and the 1.2kb target fragment neo-polyA was recovered; the transitional vector plasmid pSC65 (preservation number: CGMCC No.1097) was digested with BglII, blunted with Klenow enzyme, treated with dephosphorylase (CIAP), and the vector was recovered; the two were ligated at 16°C for 4 hours, and transformed into E. coli TOP10. Multiple single colonies were picked, a small amount of plasmid was extracted, identified by XbaI and PstI, and the correct recombinant clone was named pSC-neo.
[0050] 2. Artificially synthesized vaccinia virus vector early promoter PE6 and lacZ fusion fragment.
[0051] Genes were synthesized by overlapping PCR (Overlapping PCR). First, the ...
Embodiment 2
[0054] Example 2: Codon optimization of the nucleocapsid protein and protrusion protein coding sequence of SARS-CoV and construction of DNA vaccine:
[0055] 1. Genetic code optimization. According to the genetic codon usage frequency in the human genetic code preference table, select the most preferred genetic codon, according to the amino acid sequence of the nucleocapsid protein and protrusion protein of SARS-CoV (refer to the SARS-CoV Hong Kong strain HKU-39849 published by Genebank isolate amino acid sequence), back translated back to nucleotide sequence.
[0056] 2. Modification and adjustment of gene sequence. Small adjustments and modifications, using the degeneracy of the genetic code to eliminate redundant restriction enzyme recognition sites, eliminate potential nucleic acid secondary structures and other sequences that are not conducive to gene synthesis.
[0057] 3. Artificially synthesize the target gene. Genes were synthesized by overlapping PCR. First, the ...
Embodiment 3
[0060] Example 3: Construction of target gene expression element and vaccinia virus transfer vector
[0061] 1. PCR amplification of fusion promoter PE / L+P7.5 sequence
[0062] Design primers:
[0063] P7.5 Primer 1: 5'-GAAGATCTGTCGACTTCGAGCTTATTT-3' (SEQ ID NO: 3);
[0064] PE / L primer 2: 5'-GAGAATTCGTTTAAACCGATGC-3' (SEQ ID NO: 4)
[0065] The pE / L+p7.5PCR amplification reaction uses the kit of Dalian Bao Biological Engineering Co., Ltd., and the reaction system is as follows:
[0066] Plasmid pSC65 (the deposit number of plasmid pSC65 is: CGMCC No.1097.) 1 μl, each 1 μl of forward and reverse primers (P 7.5 primer 1, PE / L primer 2), 5 μl of 10×Pyrobest buffer, dNTP mixture (each 2.5 mM) 5 μl, Pyrobest DNA polymerase (5U / ml) 0.5 μl, ddH 2 O 37.5 μl. PCR reaction conditions: pre-denaturation at 94°C for 2min; 30 cycles at 94°C for 30s, 58°C for 30s, and 72°C for 30s; 72°C for 7min; 4°C.
[0067] The extension product of the pE / L+p7.5 PCR amplification reaction was purif...
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