CDNA Cloning,Sequence Analysis And Prokaryotic Expression Of Follistatin Gene From Guangxi Bama Mini-Pig

Follistatin(FS) is a single strand glycoprotein which is broadly distributed in vivo. As an activin-binding protein, FS involves in the animals reproductive regulating processes through the Follistatin/Activin system. Meanwhile, FS shows various biological functions on different kinds of tissues in vivo.A pair of primers was designed based on the encoding sequences of FS released at the GeneBank. The upstream primer, 5' -ATGGTCCGTCCCAAGC-3', designed from the 1 -16 nucleotides and the downstream primer , 5' -TTACCACTCTAGAATAGAAGATATAGG-3', designed from the 1008-1035 nucleotides of encoding sequence of FS were synthesized. The total RNA purified from Guangxi Bama Mini-pig ovary tissue was used as the reverse transcription template and the strands of FS cDNA was synthesized by using M-MLV reverse transcription enzyme. The full length sequences(1035bp) were amplified by the Reverse Polymerase Chain Reaction (RT-PCR) then ligated into pMD18-T vector after purification. The recombined vectors were verified through the methods of PCR, restrictive cleavage and the sequence survey, respectively. The results showed that the Bama Mini-pig FS cDNA was successfully cloned in this study. The homologous analysis indicated that the cloned BaMa mini-pig FS cDNA was highly homologous with the reported nucleotide encoding swine FS in GeneBank(99. 4%) and this gene has high conservativeness among mammalian animals (>89%).The FS cDNA sequences were amplified through another pairs of primers, which contain the restrictive enzyme sequences (BamH I and Sall). After digested, the sequences were inserted into the specific multiple cloning sites of plasmid pET 32a+ to construct prokaryotic expression vector, and transformed into Ecoli. BL₂1. After SDS-PAGE survey and Western blotting, it was comfirmed that a fusion protein was sucessfully expressed by transformed Ecoli. BL₂1 induced by IPTG (about 58KD). The result showed that the FS fusion protein was expressed in prokaryotic ce11s in the form of inclusion body and accounted for about 12% of the total protein content of the bacteria. Results from the study of influences of different induce conditions on FS fusionprotein expression indicated that the best induced temperature IPTG concentration and induce time were 37℃, 1. 0-1. 5mM IPTG, and about 4-8h, respectively.It is concluded that the Bama Mini-pig FS cONA have been cloned and the prokaryotic expression vector was constructed successfully in this study. The FS fusion protein could be successfully expressed after the IPTG inducing. These recombinant FS protein could be further applied for the developing of immunoadjuvants used in the study of biological functions of FS in swine.