Study On The Cloning And Sequence Analysis Of The Mature Region CDNA Of Inhibin Subunits For Xiaoshan Chicken, And The Fusion Expression Of Inhibin α-subunit Of Xiaoshan Chicken In E.coli

Inhibins are gonadal glycoprotein hormones comprised of a-subunit and p-subunit (PA- and pB-subunit) linked by disulfide bonds. In vivo, inhibins have biological activity in suppressing the synthesis and releasing of FSH from pituitary. The emphasis of this study is focused on cloning and fusion expression of mature inhibin a-subunit cDNA of Xiaoshan chicken in BL21(DE3) strain. The aim of the present study is to provide theoretical foundation for the effect of recombinant protein on poultry reproduction.The total RNA was extracted from follicle granulosa cells collected from chicken ovary using Total RNA Extracting Kit. The total RNA was transcripted with the reverse transcriptase to obtain the mature region cDNA sequences of Xiaoshan chicken inhibin subunits. Using pGEM-T Easy Vector and pUCm-T Vector as cloning vector, we cloned the a-subunit, pA-subunit and PB-subunit cDNA and analyzed their sequences using DNAMAN Application software and CLUSTAL W(1.60) Multiple Sequence Alignments software, compared the obtained a-subunit, PA-subunit and PB-subunit cDNA sequences with corresponding sequences of mammal and other chickens reported in GenBank. The results revealed, the mature a-subunit cDNA of Xiaoshan chicken showed 100%~98%, 71%~98%, 73%, 71%, 71%, 68% and 71% identity to that of the chickens (Xianju chicken, White Leghorn chicken and other chicken Callus gallus), cow, pig, sheep, horse, mouse and human respectively. It is 99%, 85%, 83%, 84%, 84%, 85% identical in nucleotide sequence between the PA-subunit of Xiaoshan chicken and other chickens, cow, pig, sheep, mouse and human respectively. The mature pB-subunit cDNA has 99%, 81%, 81%, 81% and 83% homology with that of the chickens, cow, pig, mouse and human respectively. And the results also indicate that, the genes of inhibin subunits are very conserved in evolution.After digesting the positive clone of the inhibin mature a-subunit cDNA, we linked it to the expressing vector pET 30a Vector cut by the same enzymes Sal I and Nco I, and constructed inhibin a-pET 30a 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 in lower temperature(30C), and purified the fusion protein by the process of repeated freezing and thawing. The SDS-PAGE electrophoresis showed that, we obtained the targeted protein. The results further indicate it is feasible to obtain a great deal of immune antigen using this method.