Cloning Of N Gene Of Vesicular Stomatitis Virus And Expression In Prokaryotic Vector

The paper has two parts. One is nucleotide sequencing of N gene of the VSV, the other is the expression of the N gene of the VSV in prokaryotic vector. The results are: 1. One pair of primers was designed according to the published sequences of VSV. The N gene of it were amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The amplified N fragments of the VSV was 1 300 bp in length by agarose gel electrophoresis. Then the fragment was cloned into the pGEM-T vector and was sequenced. Comparison on the nucleotide sequences and deduced amino acid sequences of the N gene IND wild strain,tsG31strain and HR strain showed that the homology of nucleotide sequence was 99%,and that of the amino acid sequence was 99%.These prove again that the N gene is highly conserved in VSV.2. Another pair of expression primers with digestion site EcoR I and Xho I on were designed to amplify the N genes of the VSV by PCR on the basis of the recombinant plasmid constructed in the former experiment. The recombinant plasmids pETVSV-N were obtained after being cloned pGEM-T-N1 into expression vector pET-30c(+). The insert position, the orientation and the ORF were right by PCR,digestion and sequence analysis. The 53 kDa target proteins were produced by inducing with 1.0 mmol/L IPTG. Western-blot showed that the expressed proteins could be recognized by VSV positive serum. Thus, the recombinant N protein makes the basis for establishing ELISA method and other serum method .