卵巢癌相关抗原CA125串联重复序列的原核表达纯化及抗血清制备

Prokaryotic expression and purification of a tandem repeat of ovarian cancer antigen CA125 and preparation of its antiserum

目的:建立CA125串联重复序列(CA125R)原核表达系统,表达纯化重组CA125R蛋白并制备其抗血清。方法:全基因合成一段CA125串联重复序列,并将其克隆至pET-32a(+),构建CA125R蛋白原核表达载体pET-CA125R;将构建好的pET-CA125R载体转化至E.coli BL21(DE3),确定最佳可溶性表达条件,通过Ni-NTA亲和层析系统纯化重组CA125R蛋白;纯化重组蛋白经Western blot方法验证后,免疫家兔制备抗血清。结果:成功构建CA125串联重复序列原核表达载体,确定了最佳可溶性诱导表达条件(0.5 mmol/LIPTG,15℃诱导6 h);Western blot结果证实纯化的重组蛋白正确,纯度高;制备的抗血清能特异识别重组CA125R蛋白和天然CA125糖蛋白。结论:成功建立了CA125R高效原核表达系统,并制备了高纯度重组CA125R蛋白及其抗血清。

AIM： To establish a prokaryotic expression system of the tandem repeat of CA125 （CA125R）, express and purify the recombinant CA125R protein, prepare its antiserum. METHODS： The full gene sequence of one tandem repeat of CA125 was synthesized and cloned into pET-32a（ ＋ ） to construct a prokaryoUc expression vector of the CA125R protein （pET-CA125R）. The pET-CA125R was transformed into E. coli BL21 （DE3） and the soluble expres- sion conditions were optimized; the pure recombinant CA125R protein was prepared by affinity Ni-NTA chromatog- raphy and identified by Western blotting. A rabbit was immunized with the pure recombinant CA125R protein to prepare its antiserum. RESULTS： The prokaryotic expres- sion vector of CA125R was successfully constructed. The optimal soluble induction expression conditions were 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside （IPTG） at 15℃ for 6 h. Western blotting confirmed the pure CAI25R recombi- nant protein of high purity. The prepared antiserum specific- ally recognized recombinant CA125R protein and natural CA125 glycoprotein. CONCLUSION： We successfully es- tablished the efficient prokaryotic expression system of the CA125R, and prepared the recombinant CA125R protein of high purity and its antiserum.