Synthesizing, Cloning, Expression Of Mycoplasma Suis MSG1 Gene Using PCR-based Accurate Synthesis And Overlap-extension PCR, And Its Immunogenicity Analysis

Mycoplasma suis (M.suis) were formerly classified as the rickettsial agent,Eperythrozoon suis, but comparative sequence analysis of the 16sRNA sequence, theinternational community generally reclassification it together with Mycoplasma. M.suisis a micro-organism which parasitized on the porcine red blood cells, it can cause afebrile acute anemia and icterus with low morbidity and high mortality as the maincharacteristics of blood infections diseases. In recent years, M. suis disease wasnationwide outbreak and spread of a large area of China's pig industry and caused greateconomic losses. In order to control the disease, we must make a further research in thepathogen. Now, we can search the 16sRNA gene fragment of M.suis in GenBank.Meanwhile, we can also find the other three paragraphs on the M.suis gene sequencereported in pubmed——1.8kb DNA sequence, MSG1 adhered protein, HSPA1. One ofthe 1.8kb DNA sequences of genes whether belong to M.suis is remaining controversial.Hoelzle et al. sequenced the M.suis genomic library based on the shotgun method,which has found the surface-localised protein of M. suis——MSG1. The protein MSG1was demonstrated that the sequence was homology with the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and E.coli transformants expressing MSG1 ontheir surface acquired the ability to adhere to porcine erythrocytes. And the MSG1 genehas high conserved in different isolates. So the MSG1 protein has an important functionin the M.suis.Due to the lack of the best cultivating mechanism of M.suis, which restricted to theM.suis's research, and M.suis coding genes are used in the fourth set of codon, there is aconflict with the E. coli expression system. Specifically, the M.suis encoded TGA isserine, but TGA in E. coli expression system is coded as a terminal code. Therefore, inthe prokaryotic expression system, we must use the serine codon which expressed inE.coli instead of the TGA. In this experiment, According to M. suis AM407404sequence of 1011 base pairs which GenBank has been published, and codon TGG wasreplaced by TGA, using the Graphical Codon Usage Analyser, the code which E. coli. is partial to, was substituted for the rare codes. 28 primers were synthesized, which twopairs of upper, lower specific primers contained HindⅢand NdeⅠwas designed. TheMSG1 of M. suis was synthesized by overlap PCR. The products were first inserted intovector pMD18-T. The result of sequencing showed that the products were deleted in169 and 897 which compared with the MSG1 sequences. So in order to correct theworry genes that OE-PCR primers were designed. The gene was subcloned toprokaryotic expression vecter pET28c after sequencing. The recombinant plasmidpET28c_msg1 was transformed to E.coli BL21 for expression under induction of IPTGThe result was showed that MSG1 in prokaryotic expression system can be efficientlyexpressed, and purified target protein can be obtained by electroelution. The expressedproduct was identified by Western blot and suggested that the recombinant protein hasgood antigenicity. The purified recombinant protein vaccinated to kunming mice, therecombinant protein as antigen-coated ELISA plates to detect antibodie in Kunmingmouse serum. The result was showed that MSG1 protein can be produce betterantibodies in mice.In conclusion, the study was the first cloned the MSG1 of M.suis by Synthesizingestablished the method of indirected-ELISA detecting the antibodies, analysed thesecondary structure, hydrophobicity of the genes. This will be the foundation to furtherstudy.