Gene Cloning?Expression And Anti-CDV Activity Detection Of Macaca Mulatta IFN-?

Interferon-gamma(IFN-?),produced mainly by T lymphocytes,natural killer(NK)cells,plays a critical role in anti-virus,anti-tumor,and immune modulation.It can regulate the natural and acquired immunity to the virus,and together with humoral immunity,cellular immunity,as well as some non-specific factors together with the body's defense against viral infection.In recent years,CDV has been rapidly transmitted in a large scale among animals of Macaca Lacepede in China and Japan,which leads to occurrence of severe clinical symptoms such as pneumonia in the infected animals.CDV breaks down the interspecies barrier and are transmitted from non-primate animals to monkeys of primates,which make the public health significance of CDV increasingly prominent.At present it is still controlled drug prevention and treatment of diseases based on the market there is no specific vaccines,interferon plus animals in veterinary clinical application is still in its infancy,which gave the rhesus bring distemper disease prevention a great deal of difficulty.Thus further enhance rhesus monkeys feeding management and disease control,prevention and treatment need to study new technologies and drugs,by IFN-y to improve immunity in rhesus monkeys could be one of a relatively effective rescue measures.In this study,the total RNA of PBMC of Rhesus monkevys was extracted.The IFN-gene of it was amplified by reverse transcription polymerase chain reaction(RT-PCR)and cloned into pGEM-T vector.The ORF of the IFN-y was composed of 498 nucleotides,encoding a polypeptide composed of 166 amino acids.Prokaryotic expression vector and expression strains were successfully constructed based on previously study of cloned IFN-y gene of Rhesus monkeys.Optimize the induced expression conditions of recombinant bacteria of the purified recombinant IFN-y,preclude the use of lymphocyte proliferation test and indirect ELISA assay immune activity,and to detect the cellular level recombinant IFN-y on inhibition of CDV infection.The results are as follows:1.In this study,reverse transcription-polymerase chain reaction(RT-PCR)technique,total RNA from rhesus peripheral blood lymphocytes in rhesus-?interferon gene was amplified and cloned into the vector PGEMT.By colony PCR,sequencing and sequence analysis of cloned IFN-y gene open reading frame of the 498 nuclear past acids encoding a 166 amino acid polypeptide.Comparison of the sequenced IFN-? with 11 the reported IFN-? sequence in GenBank at the nucleotide level,shared 100%identities with Macaca fascicularis Raffles,they don't have interspecific differences.2.In this study,the IFN-y gene was cloned into an expression vector pET32a(+)and constructed the recombinant expression plasmid pET32a-IFN-?,transformed into E.coli BL21(expression strain)after induced by IPTG,cell lysis for SDS-PAGE analysis,and then determine the induced expression of recombinant gene expression and the best conditions and purified prokaryotic expression products were.Optimal expression conditions were 37?,0.05 mMol/L IPTG induced 8h.The recombinant fusion protein had a nolecular weight of approximate 35.4 kD.After purification by nickel column,the target protein was harvested with a concentration of 0.2 mg/mL.3.In this experiment,the biological activity of the purified was detected by lymphocyte proliferation test and indirect ELISA assay immune activity.The result showed it can stimulate the proliferation of T cells,and has a positive correlation in a certain range,and can stimulate the secretion of IFN-gamma in peripheral blood lymphocytes of Rhesus monkeys.Real-time PCR test proved that the recombinant IFN-y at 2-flod dilition of the most obvious effect,the inhibition of CDV decreased with the doubling of dilution.In summary,the study successfly cloned Rhesus monkeys IFN-y and reconstructed prokaryotic expression vector.The recombinant protein has the high of immune activity and antiviral activity.The results of this study provide the basis for the further development of the treatment of Rhesus monkeys virus disease,and it has laid a foundation for the clinical treatment of Rhesus monkeys.