论文标题:山羊促卵泡素（FSH）基因cDNA的克隆、序列分析与表达研究
Studies of Cloning、Sequence Analyzing and Expressing on Goat Follicle-stimulating Hormone (FSH) Gene cDNAs
论文作者 张军强
论文导师 丁家桐,论文学位 硕士,论文专业 动物遗传育种与繁殖
论文单位 扬州大学,点击次数 134,论文页数 46页File Size2593k
2003-05-01论文网 http://www.lw23.com/lunwen_4474162/ 山羊;促卵泡素;基因;免疫
Goat;Follicle stimulating hormone;Gene;Immunity
利用RT—PCR技术从山羊脑垂体RNA中成功扩增出促卵泡素(FSH)α和β亚单位cDNA。山羊FSHα亚单位cDNA的阅读框由363个bp组成，编码由120个氨基酸组成的多肽，前24个氨基酸为潜在的信号肽序列，成熟肽由96个氨基酸组成，预测分子量为10.79kDa，其56和82位上存在两个潜在的N—糖基化位点。β亚单位cDNA的阅读框由390个bp组成，编码由130个氨基酸组成的多肽，前18个氨基酸为潜在的信号肽序列，成熟肽由111个氨基酸组成，预测分子量为12.53kDa，其7和24位上存在两个N—糖基化位点。山羊FSHα亚单位与已发表的绵羊、牛、水牛的氨基酸序列同源性分别为100％、97.5％和98.3％，山羊FSH β亚单位与已发表的绵羊、牛和猪的氨基酸序列同源性分别为97.7％、93.1％和93.1％。 将克隆于pGEM-T Easy质粒中的FSH β亚单位基因片段通过PCR扩增获得去信号肽的cDNA阅读框，再将此插入到原核表达载体pGEX-6p-1的谷胱甘肽S—转移酶(GST)基因的下游，获得重组质粒pGEX-6p-1-FSH β。将此重组质粒导入大肠杆菌BL21(DE3)，用IPTG进行诱导表达，通过对重组大肠杆菌菌体的超声波裂解物的SDS—PAGE电泳分析及Western-blot鉴定，结果表明重组菌能满意表达融合蛋白形式的FSH β，即FSH β蛋白与谷胱甘肽S—转移酶相连组成的蛋白质，分子量约为41kDa，与预期大小一致，电泳扫描净灰度达28％。 用粗制的GST-FSH β融合蛋白包涵体作为免疫原免疫雌性ICR小鼠，观察免疫应答反应及其产生的生物学效应。结果表明，大肠杆菌表达的GST-FSH β融合蛋白免疫小鼠制备了针对FSH β的特异性多克隆抗体。免疫组小鼠子宫的生长受到抑制，子宫重与对照组存在显著差异(p＜0.05)。由此表明，针对FSH β的多抗与小鼠体内FSH发生了免疫中和效应。 本实验研究结果，不仅揭示了山羊FSH的分子结构，而且为山羊FSH基因工程产品的研制，及开展山羊FSH免疫与应用研究奠定了基础。
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.
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