绵羊肌肉生长抑制素MSTN原核表达及其纳米抗体文库的构建与鉴定

Expression of Ovine Myostatin Gene and Construction and Identification of Nanobody Library Against Recombinant MSTN

克隆绵羊肌肉生长抑制素（myostatin,MSTN）基因并在大肠杆菌中诱导表达,纯化重组蛋白免疫健康的双峰驼（Bactrian camel）,分离其外周血淋巴细胞提取总RNA,利用RT-PCR扩增骆驼重链抗体IgG2、IgG3的可变区（VHH）基因片段,将VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1构建纳米抗体文库。结果显示,纳米抗体文库容量为9.5×10~5,挑取部分克隆进行测序分析,所获得的纳米抗体文库具有良好的多态性,为进一步筛选绵羊MSTN的高特异性纳米抗体片段奠定了基础。

The ovine myostatin gene was subcloned into the pET-32a （ ＋ ）, and transformed into E. coli BL21. The MSTN recombinant fusion protein was purified and the healthy Bactrian camel was selected for immunization with the MSTN protein. Total RNA was extracted from peripheral lymphocytes for the amplification of VHH fragments by RT-PCR. Primers designed according to the single domain gene sequences of the antibodies and the variable camel IgG2, IgG3 genes were amplified by Nested PCR. PCR products were then purified and inserted into phage mid vector pCANTAB5 E. The VHH antibody gene library was obtained by electroporating recombinant pCANTAB5E VHH vectors into E. coli TG 1 cells. The first set of antibody gene library was composed with 9.5 × 105 individual colones after cloning VHH genes and the library we possessed high diversity and good capacity ,which provides a platform for panning VHH camilid antibody aimed at ovine MSTN.