金鱼生长素Ⅰ基因的原核表达及其多克隆抗体制备  

Goldfish Growth HormoneⅠ Prokaryotic Expression and Polyclonal Antibody Preparation in Mice

作　　者：盖永锋[1] 张国广[1,2] 陈元婷[1] 陈亮[1] 

机构地区：[1]厦门大学生命科学学院,细胞生物学和肿瘤细胞工程教育部重点实验室,福建厦门361005 [2]漳州师范学院生物系,福建漳州363000

出　　处：《厦门大学学报（自然科学版）》2008年第A02期21-25,共5页Journal of Xiamen University：Natural Science

基　　金：国家基础科学人才培养基金项目(J0603649)资助

摘　　要：鱼生长激素(Fish growth hormone,fGH)是鱼类脑垂体中分泌的促进生长的单一亚基的蛋白激素.它参与鱼的生长代谢,能够加速蛋白质合成和脂类降解等生理功能,具有增强食欲、提高饲料转化率的作用.用引入酶切位点BamHⅠ和NotⅠ的引物,以质粒pBluescript SK-gfGHⅠ为模板,PCR扩增获得了gfGHⅠ编码区序列,分别定向插入原核表达载体pET-28a中,构建了原核表达载体pET28a-gfGHⅠ,pET28a-gfGHⅠ转化大肠杆菌BL21(DE3),在1.0 mmol/L IPTG(异丙基-β-D-硫代半乳糖苷)诱导下,表达出融合蛋白(His)6 tag-gfGHⅠ,该蛋白分子量大约为26 ku;经MALDI-TOF-MS以及抗HIS抗体的Western blotting鉴定,该融合蛋白即为表达的目的蛋白;经超声破碎鉴定该蛋白为包涵体形式存在,用连续超声破碎的方法,回收该蛋白纯度可达85.1%,免疫小鼠,间接ELISA法检测抗体效价为1∶1 600.该抗体的获得为gf-GHⅠ基因在酵母和水稻中的异源表达及功能鉴定奠定了基础.Fish growth hormone （fGH） produced by the pituitary cells of teleosts. It can increase the expression of protein, degradation of lipid and play an important part in the growth of fish. The gfGH Ⅰ cDNA was amplified by PCR using the oligonucleotides which were designed to generate a BamH Ⅰ site at the 5＇-end and a Not Ⅰ at the 3＇- end. The PCR. products was digested with BamH Ⅰ ＋ Not Ⅰand purified with PCR purification kit. The pure products were inserted into the corresponding BamH Ⅰ and Not Ⅰ restriction sites in the prokaryotic expression vector pET28a, and the recombinant plasmid was named pET28a-gfGH Ⅰ that possessed correct open read frame by sequence. The expression plasmid pET28a-gfGH Ⅰ was transformed into BL21 （DE3） and fusion protein （His）6 tag-gfGH Ⅰ （about 26 ku） containing a （His）6 tag at the N-terminus was expressed after 1.0 mmol/L IPTG induction. The recombinant protein was identified by MALDI-TOFMS and Western blotting. The recombinant protein was mainly in the form of inclusion body. The expressed product was purified by continuous ultrasonic wave. The purified protein was used as antigen to immune mouse directly and the results of ELISA showed that the titer of the anti-serum is about 1： 1 600. This work laid a good foundation for the function identity and the expression of fusion gene of gfGH Ⅰ in some eukaryotic cell.

关 键 词：gfGHⅠ基因 原核表达 抗体制备 

分 类 号：Q786[生物学—分子生物学]