Cloning,Expression And Immune Research Of LTB-SMS Fusion Protein

Somatostatin(SMS)is synergistic or antagonistic with growth hormone,their combined effects can constrain animal growth under species-specific level.Gene vaccine recombined by SMS gene and eukaryotic expression vector causes the animals to produce antibodies to decrease SMS and lift its inhibition of growth hormone,thus growth hormone increased and animal growth rates improved.In order to get a higher expression of the recombinant protein with 6 xHis-Tag,Escherichia coli heat-labile enterotoxin B subunit(LTB)was taking as a molecular adjuvant to enhance the immunogenicity of SMS gene and combined with somatostatin to constitute the recombinant plasmid,then the exogenous gene induced fusion protein,which lays the foundation of SMS gene engineering vaccine research and development.Antibody against somatostatin can promote the growth of animals.Because of safety and effectiveness,so far no SMS vaccine is available.Taking synthetic genes as target proteins,recombinant plasmid of pET28a vector was obtained through genetic engineering,transformed into Escherichia coli DH5a,and the right positive clones were chosen.By agarose gel electrophoresis,LTB gene molecular weight is about 350bp,synthesis of somatostatin gene molecular weight is about 100 bp.The LTB gene was combined with somatostatin gene,connected with vector plasmid pET28a,transformed into Escherichia coli BL21(DE3)competent cell,and induced the expression of recombinant proteins.Isopropyl-?-d-Galactose-Glycoside thiosulfate(IPTG)was used to induced at 37? for 4 h,with its final concentration is 1 mmol/L.Target protein was in the form of inclusion bodies in Escherichia coli,which was proved by SDS-PAGE.The NI column purification of 6×His-Tag systems were carried out to isolate,renature recombinant protein and purify,because of fusion protein with 6×His-Tag itself.8 mol/L urea dissolves collected inclusion bodies and the dosage of the imidazole elution 500 mmol/L,pillar renaturation was carried out by inclusion body purgation buffer rang from 7mol/L-6mol/L-5mol/L-4mol/L-3mol/L-2mol/L-lmol/L-Omol/L at 4?.The concentration of the recombinant protein sample after purification was 1.36 mg/mL,1.22 mg/mL,1.12 mg/mL respectively while that was 0.75 mg/mL,0.87 mg/mL,0.79 mg/mL.SDS-PAGE showed purified recombinant protein sample was a single strip.The immunological characteristics of LTB-SMS purified and determined was studied in rabbits by subcutaneous and mucosal immunization.The results showed that the serum antibody titers of LTB-SMS reached high level.It can provide substantial assistance to the construction and preparation of a vaccine,the fusion protein is expected to further develop to somatostatin gene engineering vaccine.