Studies On The N Gene Clone, Expression & Diagnosis Technique Of The Vesicular Stomatitis Virus

Vesicular stomatitis(VS) is an acute and high contagious disease for various species of mammals caused by vesicular stomatitis virus (VSV) of the family Rhabdoviridae, genus Vesiculovirus. Its particles are either bullet or cylinder shape. The sac membrane of its virion is even-distributed by many 10nm long spikes. Inside the virion, nucleocapsids are twisted tightly in a symmetrically spiral way. The VSV genome is a linear single-strand negative RNA which is about 11kb in length. The gene order of the virion RNA has been shown to be 3'…N-NS-M-G-L…5'. These five genes don't overlapped and encode N protein, P protein, M protein, G protein and L protein respectively. Among them, N protein and G protein have got great significance in immunology and diagnosis. The nuclear protein encoded by N gene is consist of 422 AA residues(47kDa), Each virion contains 1258 copies that can effectively protect viral RNA from being digested by nucleotidase. The N protein presents a group specificity, it exists in various types and subtypes of VS and plays a very important role in transcript copy. Due to its characteristics of high antigenicity , the N protein can stimulate the body producing nonneutralized antibody. In the body , as the predominant antigen, the N protein is recognized by VSV specific CTL cell and functions its role as CTL participating in immunological reaction. Being the nucleocapid protein, N protein conjugates with anterior guidance RNA forming RNA complex. Many immunological methods for testing antibody of N protein can be used to identify infections caused by VSV from those caused by vaccines.Vesicular stomatitis is a list A disease for OIE and a second class disease in China exit and entry inspection and quarantine of animal and animal products. At present, China has no VS disease. But following China entered into WTO, the international trade for animal and animal products has been increasing. It is necessary for China to develop researches on this disease to prevent it spreading to China and protect the health development of our animal husbandry.In this study , we have introduced the VSV nuclear acid from abroad and carried on the following researches. 1. Specific primer is designed on the basis of reported VSV N sequence. With VSV-IND RNA introduced from abroad, c DNA which encode the whole VSV N protein is obtained from transcription and PCR amplification. A recombinant pGEM-T-N plasmid of VSV N is established and N gene is successfully cloned into the vector of pGEM-T Easy. From sequencing, we can deduce the recombinant plasmid include complete N gene and its sequence is 1334bp in length. Through analysis of the sequence, it is proved VSV N is highly conservative.2. A specific primer is redesigned according to the result of sequence analysis. Then endonuclease sites for EcoRⅠ,XhoⅠare respectively added into the primer. Conducting RT-PCR amplification, a target gene is obtained. Cloning the gene into pET-30c, the expression vector of VSV N is successively constructed. Through analyzing the expressing protein, we find the molecular weight of the fusion protein is 53kDa in which fusion protein is about 6kDa and target protein is about 47kDa.That conclusion is consistent with the predicted size for target protein. Western blot also indicates the expression protein can produce specific reaction with the positive serum of VSV and is hopefully to become a valuable VSV diagnostic antigen. Researches on proteid reveal that VSV N gene encoding nuclear protein is a polypeptide which is made up of 422 amino acid and has a strong hydrophility. Comparing its homogeneity with that of VSV wild strains, it shows that the nuclear protein is highly conservative due to the high homogeneity of sequences which is above 98.8%. It provides a basis for establishing clinical VSV diagnostic method.3.With ultrasonic wave and lysozyme, the recombination N protein is split from bacteria. We dissolve it in guanidine hydrochloride, purify it with Ni++ affinity chromatography column and renature it in vitro. By tes...