| Mycoplasma suis (M. suis) were formerly classified as the rickettsial agent, Eperythrozoon suis, but comparative sequence analysis of the 16sRNA sequence, the international community generally reclassification it together with Mycoplasma. M. suis is a micro-organism which parasitized on the porcine red blood cells; it can cause a febrile acute anemia and icterus with low morbidity and high mortality as the main characteristics of blood infections diseases. In recent years, M. suis disease was nationwide outbreak and spread of a large area of China's pig industry and caused great economic losses. In order to control the disease, we must make a further research in the pathogen. Now, we can search the 16S rRNA gene fragment of M.suis in GenBank. Meanwhile, we can also find the other five paragraphs on the M.suis gene sequence reported in pubmed—rnpB, Inorganic phosphatase, MSG1 adhered protein, HSPA1 and ORF1,ORF2,ORF3,ORF4,ORF5,ORF6,ORF7,ORF8,ORF9,ORF10,ORF11 of M.suis. 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 MSG1 was demonstrated that the sequence was homology with the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and E.coli transformants expressing MSG1 on their surface acquired the ability to adhere to porcine erythrocytes. And the MSG1 gene has high conserved in different isolates, and proof HspA1 protein had adenosine triphosphate enzyme activity and antigenicity, but the function of rnpB, Inorganic phosphatase, ORFs of M. suis was rarely to know.Due to the lack of the best cultivating mechanism of M.suis, which restricted to the M.suis's research, and M.suis coding genes are used in the fourth set of codon, there is a conflict with the E. coli expression system. Specifically, the M.suis encoded TGA is serine, but TGA in E. coli expression system is coded as a terminal code. Therefore, in the prokaryotic expression system, we must use the serine codon which expressed in E. coli instead of the TGA. In this experiment, According to M. suis AJ504999 sequence of ORF2,ORF4,ORF5,ORF9 gene which GenBank has been published, and codon TGG was replaced by TGA, using the Graphical Codon Usage Analyser, the code which E. coli. is partial to, was substituted for the rare codes. Primers were synthesized, which two of upper, lower specific primers contained restriction enzyme was designed. The ORF2,ORF4,ORF5,ORF9 gene of M. suis was synthesized by overlap PCR. The products were first inserted into vector pMD18-T. The result of sequencing showed that the products were deleted in 518 which compared with the ORF2 sequences. So in order to correct the genes that OE-PCR primers were designed. The gene was subcloned to prokaryotic expression vecter pET28a or pET32a after sequencing. The recombinant plasmid p PET-28a/ORF2,PET-28a/ORF4,PET-32a/ORF5, PET-32a/ORF9 were transformed to E. coli BL21 for expression under induction of IPTG. The result showed that the protein was obviously appeared by SDS-PAGE and Western-blot. At the same time,the target protein of secondary structure, 3D structure and epitope were predicted with bioinformatics software.In conclusion, the study was the first cloned the ORFs gene of M.suis by synthesizing, analysed the secondary structure, 3D structure and epitope of the genes. This will be the foundation to further study.
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