Construction Of Eukaryotic Plasmid PVAX1-SPG And Expression In Mammalian Cells

Dental caries is a contagious disease whose major pathogenic bacteria are Mutans Streptococcus, including Streptococcus mutans (S.mutans) and Streptococcus sobrinus (S.sobrinus). Surface protein antigen (SpaP) and glucan-binding proteins (Gbp), considered as important virulence factors, involved in the adhesion of streptococcus mutans in the teeth. SpaP and Gbp have good immunogenicity, thus are able to induce protective immunity.The purpose of this study is to construct and identify eukaryotic plasmid expression pVAX1-SPG by inserting gene spap-p and gbd, which are acquired from Streptococcus mutans UA159, multiple cloning sites of pVAX1. The recombinant plasmid was transfected into mammalian cells for the observation of protein expression. Our results will provide the material basis for further study on gene vaccine against dental caries.Method:The study consisted of two parts:Part 1 The construction of recombination eukaryotic plasmid pVAX1-SPG. It is constructed by PCR amplification of spap-p gene of Streptococcus mutans'surface protein SpaP and gbd gene of glucan-binding proteins. The target gene was inserted into cloning sites of eukaryotic expression vector pVAXl for construction of the recombine eukaryotic plasmid pVAX1-SP and pVAX1-GG. After the inserted gene checked by digestion of restriction enzymes KpnI&EcoRI, primers were designated. Gene spap-p and gbd were inserted into multiple cloning sites of eukaryotic expression pVAX1 to construct the recombine eukaryotic plasmid pVAX1-SPG. Data was analyzed through Dual enzyme and gene sequence checking method. Part 2 Recombine eukaryotic plasmid pVAX1-SPG expressed in the transfected 293T cells:Transfect the 293T cells with recombine Eukaryotic plasmid expression pVAX1-SPG. SABC will be used to test the protein expression.Result:(1) Through PCR amplification the gene of Streptococcus mutans Surface protein SpaP and glucan-binding proteins GbpA, spap-p(1.2kb), gbd(1.18kb) were obtained. Through dual enzyme digestion with KpnI/EcoRI, the recombine Eukaryotic plasmid pVAX1-SP showed 3.0kb and 1.2kb two markers after electrophoresis. Through digestion of restriction enzymes KpnI&EcoRI, the recombine Eukaryotic plasmid pVAX1-GG showed 3.0kb and 1.18kb two markers after electrophoresis. Through dual enzyme digestion with KpnI,EcoRI, the recombine eukaryotic plasmid pVAXl-SPG showed 3.0kb and 2.4kb two markers after electrophoresis, which was consistent with designed. After sequence test and comparison, nucleotide in the recombine eukaryotic plasmid pVAXl-SPG has a 99% homology as the source. The gene was inserted in the correct location and the reading frame was not changed.(2) After transfect the 293T cells with recombine eukaryotic plasmid pVAX1-SPG, experimental group had brown products in cytolymph which was different from the control group significantly.Conclusion:The recombine eukaryotic plasmid pVAX1-SPG by inserting spap-p(gene of Streptococcus mutans' Surface protein SpaP P region) and gbd (gene of glucan-binding proteins) were successfully constructed. The interest protein could be correctly expressed after transfecting the recombine eukaryotic plasmid expression pVAXl-SPG into the 293T cells.