Prokaryotic Expression And Preparation Of IHNV-G,and The Application Of The Antibodies In IHNV Detection

Infectious hematopoietic necrosis(IHN)is often found insalmon and trout in science,and it is extremely easy to cause the occurrence of acute viral deaths in trout,which is a kind of serious infectious disease.The mortality rate of IHN is high,especially for fry great harm.Studies show that infectious haematopoietic necrosis virus(IHNV)genome is composed of single negative stranded RNA.IHN is an enveloped virus,and possesses a external glycoprotein G inserted into the viral membrane responding for attachment to the membrane of host cell,and help virus get entry into the host cell.We retrieved 59 gene of IHNV-G from Gene Bank.After comparison,conservative codons were selected to optimize the overall,at the same time,keeping the amino acid sequence invariable and reading frame still right.And restriction enzyme cutting site sequence of KpnI and SalI were added to the both ends,besides,his tag was added to the N-terminal.Finally,a 1545 bp IHNV-G protein was formed.We designed and synthesized the gene sequences of IHNV-G protein according to the Gene Bank and connected it with pET-30 a to construct recombinant plasmid pET-30a-IHNV-G.After PCR,restriction enzyme digestion and sequencing validation,transformed it into E.coli BL21(DE3)plySs and induced it to express the target gene,confirmed the expression of IHNV-G protein by Western Blot.We studied the optimum fermentation condition from two aspects including time,and IPTG concentration.Thallus were broken by ultrasonication,then collect the supernatant and the precipitation to determine the expression form of the IHNV-G protein.The collected inclusion bodies were washed,dissolved,renatured,we obtained the IHNV-G after desalting and freeze-dried.The protein was used to immunize rabbits to acquire the polyclonal antibody,and then establish a rapid method of indirect-ELISA.Results showed that the prokaryotic expression plasmid pET-30a-IHNV-G was successfully constructed.After induction,the positive recombinant strain expressed the IHNV-G protein successfully.The expression conditions of 37 ?,1 mmol / L IPTG and fermentation time 4 h were optimal.The IHNV-G protein expressed in the form of inclusion bodies,after separation and purification,average of 1 Liter fermentation broth would collect 7 g thallus and 0.2-0.3 g protein powder.The titer of anti-serum of IHNV-G and IHNV reached to 1:32000 and 1:16000 respectively,moreover,the ratio of cross-reaction with other virus was 0%,and ELISA results was in accordance with PCR in a ratio of 98%.Conclusion: The method of indirect-ELISA in this research had good specificity and sensitivity,and it was suitable for rapid detection and epidemiological investigation of IHNV infection in trout.