Prokaryotic Expression Of ORF68 Partial Gene In KHV And Its Monoclonal Antibody Preparation

Koi herpes virus disease has caused severe financial losses in common and koi carp culture industries worldwide,This phenomenon is mainly caused by there no fast,safe and effective diagnostic methods,and can not be found to diagnosis,it can be diagnosed early in the early detection of problems in time to solve the problem by application of monoclonal antibody technology.Monoclonal antibody technique in koi herpes virus disease starts late.There are only three related reports abroad about it,and there have been no properly documented successes,so this study is dedicated to screening monoclonal antibody of koi herpes virus,it`s promote carp healthy growth,for China’s carp breeding industry is of great significance.The ORF68 gene fragment of 753 bp,897 bp and 1500 bp was inserted into the p MD18-T vector by PCR amplification using the KHV-CJ strain,Which was isolated and preserved by our laboratory.The cloning plasmids p MD18T-753,p MD18-T-807 and p MD18-T-1500 were constructed successfully by PCR and double digestion.By BLAST,the homology of the obtained gene fragment could reach 100%.The recombinant plasmids pET32a-753,pET32a-807 and pET32a-1500 were transformed into BL21 expressing bacteria by IPTG,and the recombinant protein was analyzed by IPTG.The results showed that the recombinant protein could be present in the inclusion bodies of Escherichia coli and the soluble form at the same time.The molecular weight was 45.61 KDa,47.59 k Da and 73 k Da.With His Trap HP for purification,The concentration of protein was calculated as follows: recombinant protein p ET32a-753: 0.46 Mg / ml,recombinant protein p ET32a-807: 0.57 mg / ml,recombinant protein p ET32a-1500: 0.51 mg / ml.The mice were immunized with the immunization program and the serum titer of the mice was measured by ELISA.The spleen cells of the immunized mice were fused with SP2/0 cells.After screening with HAT conditioned medium,the supernatant titer of the culture medium was determined.After the positive clones were screened for several times,the cells with high expression were screened according to the OD450 nm value,and limiting dilution assay,appositive monoclonal cell was chosen,the cells were collected to prepare ascites,then its subtype,reactionogenicity were tested.As a result,A total of 227 positive monoclonal cell lines were selected by screening the supernatant of monoclonal cell culture supernatant,and 15 positive results were screened after repeated detection.Five higher OD450 nm values were selected according to the OD450 nm value,named: 807-1D3,807-1D6,807-3E5,1500-3D2,1500-4G2,the final screening of a more stable positive strains: 807-1D6,it belongs to Ig G1,and it has good reactionogenicity with recombinant protein p ET32a-807 by Western-blot.