Cloning And Prokaryotic Expression Of The Prolactin Coding Sequence In Zi Goose And White Goose

Pituitaries of three Zi Gooses and three White Gooses were used to study the cloning, prokaryotic expression and immunogenicity of prolactin by molecular biology methods. The total RNAs were extracted from pituitary collected from Zi and White Goose by Trizol Total RNA Extracting method. The total RNAs were transcripted by the reverse transcriptase into cDNA sequence. With reference to the Wanxi White Goose prolactin gene in GenBank database, a pair of DNA primers were designed and synthesized. Two 690 bp fragments containing prolactin gene of Zi and White Goose were successfully amplified by RT-PCR and cloned into pMD18-T vector plasmid, enzyme digested by BamHI and HandⅢand analyzed their sequences with DNAstar Application software, compared the obtained prolactin cDNA sequences with corresponding sequence of Wanxi White Goose reported in GenBank. The results revealed that the mature prolactin cDNA of Zi and White Goose had 99.57 % homology that of Wanxi White Goose, and the results also indicated that there were three nucleotides different from Wanxi White Goose cDNA sequence, and in the sequences of pMD18-T-PRL of Zi and White Goose contain all the coding sequence for mature protein of prolactin gene.After digesting the positive clone of the prolactin mature cDNA, they were linked to the pET-32a (+) Vectors digested by the same enzymes, BamHI and HandⅢ, and constructed transformed prolactin-pET-32a (+) expression vector and screened recombinant, then transformed it into BL21(DE3) strain. Induced the expressing strain BL21(DE3) by adding IPTG into the medium to produce the protein of interest constructed lower temperature (37℃), and inclusion body protein were made. Tricine-SDS-PAGE showed that the recombinant proteins had molecular weight approximately to 45 kDa. The recombinant prolactin genes were expressed efficiently in form of inclusion body.Prolactin genes of Zi and White Goose were constructed into pET-32a (+)-PRLs. And pET-32a (+)-PRLs were expressed in the E.coli. expression system. The results laid a solid foundation for the future of prolactin in goose.Purified fused proteins and digested PRL monomers by thrombin were injected into Kuming mouse, the results showed that the fused proteins of PRL can be tested clearly, and both of fused proteins and monomers have efficient immunogenicity.