Constructing The Expression Of Fusion Protein And Polyclonal Antibody Through SARS Coronavirus Spike Protein Rbd Gene Fragment

ObjectiveThe objective of our study is to construct the basis of HIS fusion expression model through amplifying SARS-Cov S protein RBD gene and fusing with PET-32(a) vector to perform the prokaryotic expression.It will be the supportment of searching spike protein and SARS prevention, prognosis diagnosis, treatment and so on.Methods1, Constructing of PET32(a)-RBD plasmid and performing prokaryotic expression.PET32(a) vector and RBD fragment were combined by high-efficiency T4 joinase, which will be cultured in competence BL21 E.Coli after placing. The plasmid was acquired and digested before the Agar Gel Electrophoresis. In order to confirm the plasmid combination, the plasmid was sent to detect the sequence. After that, the plasmid was re-tranfected to BL21 E.Coli to induce the protein expression, with control group parallel. The virus stains were be kept carefully.2, The polypeptide synthesis of SARS-CoV S1 protein and construction of the related antibodies.We analyzed primitive sequence of S protein RBD fragment with computer. Three hydrophilia peptide was translated, through which we acquired three immunogenic protein: KLH-CQ19, KLH-LV14 and KLH-TY11. Use it Immuning the BABL/C mice. After 10 days of third injection, ELISA was performed on blood test. The result was compared to the original blood preserved as the negative control group from the tail before the first injection.Result1, The construction of PET32(a) vector combination and fusion protein expression.(1) Gel recovery PET32(a) and RBD fragment after digesting: The recovery products was compared to PET32(a) and RBD fragment band through Agar Gel Electrophoresis.(2) Fusion protein expression under IPTG induction: the positive plasmid was cultured and expressed under IPTG induction. Significant difference could be observed by comparing the IPTG induced group and un-induced one.(3) The result indicated the HIS system was constructed completely.2,Polypeptide antibody synthesis(1) Analysis spike protein: The computer told us the immunogenicity and hydrophilic of polypeptide CQ19, TY14 and LV11.(2) Acquired three polypeptide antibody: Synthesis polypeptides was comfirmed by amino acid analysis apparatus.(3) KLH-CQ19 mice resistant to polyclonal antibody: ELISA results showed that the serum antibody production was weakly positive. Titer of 1:103 and 1:104 is about 2 times of negative control OD value.(4) KLH-LV11, KLH-TY14 immunized mice had no antibody production.Conclusion1, This study utilized fusion protein technique to combine the SARS-CoV Spike protein RBD fragment and PET32(a) vector, constructing the PET32(a) fusion platform on our lab, which was the basis of the further experiment on spike protein. The high-quality vectors we used on this study gifted the advantage on testing and depuration of fusion plasmid.2, This study utilized the gene analysis technique on building the high-immunogenity antigen-polypeptide fragment, through which we acquired highly purified antibody through the BABL/c marine model. The result of ELISA showed up the high-efficiency CQ19 antibody could be acquired through this technique, which provided the possibility on providing the SARS vaccine on clinical usage.