Clone And Expression Gene Of Hexon Of Infectious Canine Hepatitis Virus

The Infections Canine Hepatitis (ICH) caused by the Infections Canine Hepatitis Virus is an common acute septicemic infectious diseases. Its mainly hosts is Canines and Foxs. This disease is one of the most hazardous animal plague in Canines and Foxs farms. The major characteristic of this disease are saddle type fervens, severity harmful hemagglutination, damaging liver and cornea cloudiness in the clinic.This disease principal happens in puppy. The mortality can reach up to 40%. So it causes large loss in canines breed industry. The research job concludes three parts that were listed after this paragraph.1.Cloning and sequence analysis of the infections Canine Hepatitis Virus Hexon gene The specificity primer was designed through Infections Canine Hepatitis Virus Hexon gene sequence released in GenBank. The whole was abstracted from the blood of the sick canines. Hexon gene was cloned by PCR through the genome. The it was cloned into a vector to combine a recombing vector. After laboratory identification, the recombing vector was sequenced. The result show that the whole length of the sequence of the cloned Hexon gene is 2718bp. It encode a 126kDa protein which has 905 amino-acid residues. The cloned Hexon gene has six mutations. The sequence of cloned Hexon gene was tanslated into protein. Then we found that the mutations in 9bp,2664bp and 2712bp are nonsense mutation,they do not cause changing of amino acids.The mutations in 686bp has changee the code of amino acid—from Val to Ala.The mutations in 1678bp has changed the code of amino acid—from Ile to Leu. The mutations in 2236bp has changed the code of amino acid—from Phe to Leu.2.Prokaryotic expression of Infections Canine Hepatitis Virus Hexon gene and its immunological activity detection. The recombing Prokaryotic expression plasmid pGEX-6p-1-Hexon has been constructed. And multicopy recombinant strain was obtained by transforming pGEX-6p-1-Hexon to E coli with CaCl2. Then the E.coli was induced by IPTG to express the recombing protein. The recombing protein was analyzed by SDS-PAGE and Western-Blot. The results shows that the 126 kDa protein that was expressed by the positive strain through IPTG inducing has immunological activity.3.Optimization of Prokaryotic Expression of Infections Canine Hepatitis Hexon Gene and the purification of the recombing proteinIn order to improve the expression level of Infections Canine Hepatitis Hexon gene in Escherichia coli, the effects of various expressing conditions including culture temperature ,inducing time, and the final concentration of inductor IPTG on the expression was researched.. Then the the best condition of expression was decided with the results of the experiment. The strain Bl21(DE3) should be cultured in 37℃with 2.25h, then using the 2 mmol/L IPTG as final concentration to induce expression for 8 h at 27℃..The Infections Canine Hepatitis Hexon Gene was cloned and expressed very successful in this study. The fusion protein was expressed in high level and quality through the optimization of expressing condiction. It is provid the basement of studing mechanism of action of Hexon protein and exploring subunit vaccine.