狮头鹅PRL基因的克隆及原核表达

畜牧兽医学报 ›› 2011, Vol. 42 ›› Issue (3): 343-348.doi:

狮头鹅PRL基因的克隆及原核表达

贾汝敏¹*，吴慧英¹，刘铀²，张丽¹

1.广东海洋大学动物科学系，湛江524088；2.广东海洋大学动物医学系，湛江524088

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2011-03-25 发布日期:2011-03-25
通讯作者: 贾汝敏

Cloning and Prokaryotic Expression of PRL Gene in Shitou Goose

JIA Ru-min¹*,WU Hui-ying¹,LIU You² ,ZHANG Li¹

1.Department of Animal Science, Guangdong Ocean University, Zhanjiang 524088, China;2.Department of Veterinary, Guangdong Ocean University, Zhanjiang 524088, China

Received:1900-01-01 Revised:1900-01-01 Online:2011-03-25 Published:2011-03-25

摘要/Abstract

摘要： 为了进一步研究PRL对狮头鹅繁殖性能的生理调节机制，采用RT-PCR方法从狮头鹅脑垂体组织扩增获得PRL基因的cDNA 序列，克隆至pMD18-T载体获得重组质粒pMD18-PRL，并进行序列测定和分析。将测序正确的cDNA 序列定向克隆到pET32a (+)，构建表达载体pET32a-PRL，并转化至BL21(DE3)大肠杆菌表达目的蛋白，经IPTG 诱导后进行SDS-PAGE检测和Western Blot分析。狮头鹅PRL基因编码区含有600个核苷酸，编码199个氨基酸（GenBank No. GQ856665），与其它禽类PRL具有高度保守性；狮头鹅PRL蛋白二级结构由多个α螺旋和β转角及无规卷曲构成，推测N端70~76、95~102、150~155和207~213区段为其抗原表位的优势区。SDS-PAGE检测表明狮头鹅PRL基因在大肠杆菌中获得了高效表达，可溶性表达产物约占全菌总蛋白的53.6%，融合蛋白分子量约41 ku，并用镍柱亲和层析法分离纯化融合蛋白。Western Blot 分析证实了所获的融合蛋白具有较强抗原性。试验结果为进一步研究PRL基因的生物功能及其对狮头鹅就巢、产蛋等生产性状的影响奠定了基础。

Abstract: To further study the physiological regulatory mechanisms of PRL on the reproductive performance of Shitou goose. The cDNA of goose prolactin (PRL) gene was amplified from anterior pituitary gland of Shitou goose by RT-PCR. Then the PCR product was cloned into the pMD18.T vector to construct the pMD18-PRL for sequencing. And then the cDNA was subcloned into the prokaryotic expressing vector pET32a (+). Subsequently, the recombinant plasmid pET32-PRL was transformed into the E-coli BL21 (DE3) expression bacteria, then the recombinant PRL was induced by IPTG. The purified recombinant PRL was detected by SDS-PAGE and Western Blot. The results showed that coding region of PRL gene was comprised of 600 nucleotides（GenBank No. GQ856665） and encoded 199 amino acids putative protein which shared highly conservation with that of other birds. It was found that PRL protein was comprised of several α-Helixes, β-Sheets and Coils. It was inferred that the superiority region of antigenic epitope were in the N′ 70-76，95-102，150-155 and 207-213 sections. A high proportion of soluble PRL fusion protein about 53.6% of total bacterial protein was obtained in E.coli BL21 (DE3). The molecular weight of the fusion protein was about 41 ku. The Western Blot result showed that the recombinant protein was recognized by antiserum specifically. The result of this study will provide basis for further study of the biological function of prolactin protein and the effects of PRL on the broodiness and production traits of Shitou goose.

贾汝敏;吴慧英;刘铀;张丽. 狮头鹅PRL基因的克隆及原核表达[J]. 畜牧兽医学报, 2011, 42(3): 343-348.

JIA Ru-min;WU Hui-ying;LIU You;ZHANG Li . Cloning and Prokaryotic Expression of PRL Gene in Shitou Goose[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2011, 42(3): 343-348.