罗非鱼AMH基因的原核表达及多克隆抗体制备

Cloning and expression of anti-mullerian hormone partial gene and preparation of polyclonal antibody

抗苗勒氏管激素(anti-mullerian hormone,AMH),也称苗勒氏管抑制物质(mullerianin hibiting substance,MIS),为肽类生长因子,属于TGF-β生长和分化因子家族。为研究AMH对奥利亚罗非鱼性腺发育的作用,应用DNAstar软件分析罗非鱼AMH基因的抗原性,选择抗原性较强的22～243氨基酸作为目的片段构建了AMH的原核表达载体并进行融合表达。首先利用RT-PCR方法从性腺中扩增出长约663bp的目的序列AMH基因,克隆至T载体中,经酶切鉴定和序列测定分析确认序列的正确性后将此片段克隆到表达载体pGEX-5x-1中构建重组表达质粒pGEX-AMH,并在大肠杆菌BL21中获得了高表达,目的蛋白约占菌体总蛋白的38.7%。菌体经溶菌酶裂解,制备无细胞抽提液,GSTrap FF column柱层析后得到分子量为49ku单一条带的目的蛋白。目的蛋白经FactorXa酶切裂解,GSTrap FF column过柱纯化后得到纯化的AMH蛋白,分子量为26ku,浓度为2.6mg/mL。以每只20μg的剂量4次免疫ICR小鼠,免疫小鼠可以检测到特异性针对AMH蛋白的血清抗体应答,免疫组抗体水平显著高于空白组(P<0.05),且加强免疫第5周后抗体效价为0.672±0.411,达到高峰值,血清效价为1∶2000。试验结果表明表达产物具有免疫原性,可以刺激机体产生免疫应答。

Anti-mullerian hormone （AMH）,（also called mullerian-inhibiting substance,MIS）,a member of the transforming growth factor-β family （TGF-β）,is a peptide growth factor,which is the regression of the Müllerian duct in the male foetus during early testis differentiation. To study the function of the AMH protein and the distribution of AMH in differentiating tilapia testis and ovary,the partial AMH cDNA was cloned using reverse transcription polymerase chain reaction （ RT-PCR） and expressed in E. coli and polyclonal antibody was prepared for the further study. The antigenicity of AMH was first predicted by using DNAstar software in which the 22 - 243 amino acids have strong antigenicity and immunogenicity. at the same time, the special primers were designed by PRIMER 5. 0 and the cDNA encoding AMH was amplified from total RNA of O. aurea gonad by RT-PCR,and blasted against other AMH cDNA sequences in the GenBank. The analysis of the sequence data indicated that the coding region of the cDNA fragment,which encoded 221 amino acid residues,was about 663 bp in size. The amplified cDNA fragment was cloned into the prokaryotie expression vector,pGEX-5x-1,to produce the expression vector pGEX-AMH. The recombinant plasmid was transformed into E. coli BL21. AMH-GST fusion protein was obtained after the addition of IPTG into the growth media. SDS-PAGE analysis revealed that the AMH-GST was expressed after induction with IPTG for 4h. A protein band of 49 ku appeared on SDS-PAGE gel and was proved by Western blot. The mass production of the recombinant protein was about 38. 7% of total bacteria protein. After purification and cleavage of the fusion protein,purified AMH protein could be obtained. Then the fusion protein was used to immunise some ICR mice to produce anti-AMH antibody. This fusion protein could significantly elicit specific antibody response in immunized mice compared with the blank groups,and the peak of serum reached 0. 672 ± 0. 411 at the 5th week after immunization. These results demonstrated that recombinant protein could induce high AMH antibody responses in laboratory animals. In this study,we report the expression of partial AMH gene and preparation of polyclonal antibody of recombinant AMH protein. For nearly two decades,research on AMH had been focused almost exclusively on mammals and birds. There were no reports of an AMH orthologue in teleost fish,and there might even have been some doubts about its existence given the name of this hormone and the fact that modern teleost do not have Müllerian ducts. Our study first reports that an AMH exists in tilapia and the polyclonal antibody was raised. In present study,the GSTrap FF column was used to purify the recombinant protein and the high purity AMH protein was obtained. Prokaryotic recombinant protein expression systems have several advantages. These include ease of culture,and very rapid cell growth. Expression can be induced easily in bacterial protein expression systems using IPTG. Also,purification is quite simple in prokaryotic expression systems and there are a lot of commercial kits available for recombinant protein expression. In conclusion,our results showed that partial AMH gene was cloned and polyclonal antibody was prepared for further research.