河南华溪蟹2种C型凝集素原核表达及多克隆抗体的制备

Prokaryotic Expression of Two C-type Lectins from the Freshwater Crab Sinopotamon honanense and Preparation of Polyclonal Antibodies

C型凝集素是一类模式识别受体蛋白,在先天性免疫中发挥重要作用。为了深入探明河南华溪蟹(Sinopotamon honanense)2种C型凝集Sh Lec21和Sh Lec23在先天性免疫中的作用,研究将Sh Lec21和Sh Lec23基因部分编码区序列连接至p GEX-4T-1,构建了重组表达载体p GEX-4T-1-Sh Lec21与p GEX-4T-1-Sh Lec23。随后将重组表达载体转至BL21(DE3)感受态细胞,经异丙基-β-d-硫代半乳糖苷(Isopropyl-β-d-thiogalactoside,IPTG)诱导,表达重组Sh Lec21-GST与Sh Lec23-GST融合蛋白并纯化。再以纯化蛋白作为抗原免疫日本大耳兔,第4次免疫后分离抗血清,并采用ELISA法检测抗体效价。结果显示,在28℃,0.2 mmol/L IPTG,诱导10 h条件下,可获得Sh Lec21-GST和Sh Lec23-GST重组融合蛋白以包涵体形式高效表达;制备的兔抗河南华溪蟹Sh Lec21-GST与Sh Lec23-GST多克隆抗体效价均在1∶100 000以上。研究成功对河南华溪蟹Sh Lec21与Sh Lec23进行了原核表达,首次制备了高效价的抗河南华溪蟹Sh Lec21与Sh Lec23多克隆抗体,为后续研究Sh Lec21与Sh Lec23基因在河南华溪蟹先天性免疫中的作用提供了有力的工具。

C-type lectins are a class of pattern recognition receptor proteins that play an important role in innate immunity. In order to further understand the role of two C-type lectins, Sh Lec21 and Sh Lec23 from Sinopotamon honanense, on invertebrate immunity, some coding regions of Sh Lec21 and Sh Lec23 genes were linked to p GEX-4 T-1 to construct a recombinant expression vector p GEX-4 T-1-Sh Lec21 and p GEX-4 T-1-Sh Lec23. Subsequently, the recombinant expression vector was transformed into BL21（DE3）competent cells and induced by Isopropyl-β-d-thiogalactoside（IPTG）to express recombinant Sh Lec21-GST and Sh Lec23-GST fusion protein. The purified protein was used as antigen to immunize Japanese big-eared rabbits. The antiserum was isolated after the four immunization and the antibody titers were detected by ELISA. The results showed that Sh Lec21 and Sh Lec23 recombinant fusion proteins were efficiently expressed as inclusion bodies in the presence of 0.2 mmol/L IPTG at 28 ℃ for 10 h. The antibody titer of the polyclonal antibodies against Sh Lec21-GST and Sh Lec23-GST were all above 1∶100 000. In summary, we successfully carried out prokaryotic expression of Sh Lec21 and Sh Lec23 in Sinopotamon honanense, and the first high-titer anti-Sh Lec21-GST and anti-Sh Lec23-GST polyclonal antibodies, which provided a powerful tool for a follow-up study on the role of Sh Lec21 and Sh Lec23 genes on the innate immunity of Sinopotamon honanense.