Cloning Of Sus Scrofa Reg3 And Reg4 Gene And Prokaryotic Expression Of Reg4 Variant2 In E.coli

Reg3 and Reg4 are the new members of regenerating islet-derived famify{REG&Reg). Regeneration gene family (regenerating islet-derived family, REG. or Reg) be isolated from rat isolated islets of the cDNA library by Terazono regeneration in 1988. Reg family are calcium-dependent lectin superfamily (C-type lectin super family).They encode secreted proteins who can stimulate the growth of pancreatic p cells. Today, we kown Reg protein may not only controlβcell regeneration, but also play important roles in other mammals'physiological and pathological activities, such as they can be used in the proliferation, anti-apoptosis of liver, gastrointestinal tract, pancreas cells, In addition, they also related to diabetes, inflammation and trauma. According to the protein structure, Reg family can be divided into four subtypes: Regl, Reg2, Reg3 and Reg4. The Reg family of a structure and three-dimensional structure are similar and the four members are conserved in evolution by compared protein sequences with bio informatics software.To investigate if Reg play the same roles in the Sus scrofa, we cloned Sus scrofa Reg3, Reg4 gene and obtained cognate fusion protein. The study was divided into two series.I . Clonings Sequence Analysis and structure prediction of Sus scrofa Reg3, Reg4. A pair of cloning primers were designed according to the cloning principle of homologous sequence. Total RNA extracted from Sus scrofa intestinal tissue was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into E.coli JM109 competent cells. The positive clone was identified and the sequence was sequenced, analyzed and structure prediction. The Sus scrofa Reg3 and Reg4 gene were successfully cloned in present study, the sequences have been submitted to GenBank(Accession FJ531494, FJ469910, FJ469911). And we detected they are in the tissue distribution initially.II. Prokaryotic Expression of Sus scrofa Reg4 Variant2 in E.coli a pair of expression primers were designed according to the cloned sequence. A fragment of Sus scrofa Reg4 Variant2 cDNA containing EcoRI/HindIII was amplified from recombinant vector by PCR. The fragment digested by EcoRI /HindIII was subcloned into pET21b(+)vector to construct a recombinant prokaryotic expression vector, pET21b(+) ~ Reg4 Variant2, the recombinant vector was transformed into E.coli BL21 competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The prokaryotic expression vector pET21b (+)~ Reg4 Variant2 was successfully constructed. The fusion protein expressed in E.coli BL21 was about 19.23kDa and mainly existed as inclusion bodies. It provides the experiment basis for further researching its biological function.