Studies Of Cloning,Sequence Analyzing And Expressing On Goat Follicle-stimulating Hormone (FSH) Gene CDNAs

The cDNAs for goat follicle stimulating hormone (FSH) a- and P-subunit were amplified from the anterior pituitary RNA using RT-PCR technique. The open reading frame (ORF) of the a-subunit cDNA encoded a precursor protein of 120 amino acids, of which the first 24 amino acids were predicted to be the signal peptide. The mature peptide had a predicated molecular weight of 10.79 kDa and two potential AMinked glycosylation sites at positions 56 and 82. The P-subunit cDNA encoded a precursor protein of 130 amino acids, of which the first 18 amino acids were predicted to be the signal peptide. The mature peptide had a predicted molecular weight of 12.53kDa and two potential AMinked glycosylation sites at residues 7 and 24. The amino acid sequence of the goat FSH a-subunit was 100%, 97.5% and 98.3% identical to that of sheep, cattle and water buffalo, while the goat FSH P-subunit was 97.7%, 93.1% and 93.1 % identical to that of sheep, cattle and swine.The cDNA for goat FSH p-subunit was amplified from recombinant plasmid pGEM-T-FSHβ using PCR technique, and then transcloned into the prokaryotic expression vector pGEX-6p-1. FSH β gene was genetically inserted at the downstream of the 3'-terminus of the gene encoding for enzyme glutathione S-transferase, which served as a carrier in this expression system. Recombinant expression plasmidpGEX-6p-1-FSH β was constructed, and transferred into E. coli BL21. After induced with IPTG, FSH β gene was expressed as a fusion protein linked with the GST designated as GST-FSHβ. The results of SDS-PAGE and western-blot assay demonstrated that the protein expressed by the recombinant plasmid pGEX-6p-1-FSHβ can specifically response to the antiserum against GST antibody. These indicate that theexpected fusion protein with a molecular weight (MW) about 41 kDa was successfully obtained in E. coli. The fusion protein was detected up to 28% of the total bacterial protein expressed.The crudely isolated inclusion bodies, including GST-FSHP fusion protein, were applied to immunize ICR female rats for observing the FSHβ-induced immune reaction and biological effects. The results suggested that the rats immunized with GST-FSH β fusion protein can elicit a specific anti-FSH β polyclonal antibody. The uterine growth of the experimental rats was suppressed, so that the uterine weight of the experimental rats was significantly different from those of the controls (p<0.05). These showed that the polyclonal antibodies produced in rats following immunization with GST-FSHβ fusion protein could bind to and immunoneutralize heterodimeric rat FSH in vivo.These results all gained above not only discover molecular structure of goat FSH, but also lay the groundwork for the research of genetic engineering product, immunity and application of goat FSH.