Ju Jun, Li Xin-li, Wang Wan-xia, Xu Xiang-hong, Ma Ya-ru, Li Ping, Xie Zhen
Clinical Laboratory Center, Gansu Provincial People's Hospital, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 Jun;26(6):563-5.
To purify recombinated GNLY which is expressed by prokaryotic expression system, and to prepare the monoclonal antibody (mAb) against it.
The solvable protein was purified by affinity chromatography. And employing the fusion protein GNLY immuned BALB/c mice, using conventional hybridoma technology prepared the mAb against human GNLY. Then purified and determined for titer by indirect ELISA method, for antigenic epitopes by additive ELISA, for relative affinity index by sulfocyanate elution method, and analyzed for subclass, specificity and stability.
We got the soluble reactive fusion GNLY, and its purity and content were 95%, 0.8 g/L, respectively. Four cell strains secreting mAb against human GNLY were screened, 6C8, 9C6, 5G7and5E5. Their neutralizing titers were 1:100-1:3 200 and (0.1 - 8) x 10(-4) in supernatant and ascites.
We successfully purified the fusion protein of GNLY, and prepared the mAb against human GNLY, lying a certain foundation for its laboratory and clinical research.
纯化原核表达系统表达的重组颗粒溶素（GNLY），并制备抗其单克隆抗体（mAb）。
通过亲和层析纯化可溶性蛋白。用融合蛋白GNLY免疫BALB/c小鼠，采用常规杂交瘤技术制备抗人GNLY的单克隆抗体。然后通过间接ELISA法纯化并测定效价，通过竞争ELISA法分析抗原表位，通过硫氰酸盐洗脱法测定相对亲和指数，并分析亚类、特异性和稳定性。
获得可溶性反应性融合GNLY，其纯度和含量分别为95%、0.8g/L。筛选出4株分泌抗人GNLY单克隆抗体的细胞株，分别为6C8、9C6、5G7和5E5。其在上清液和腹水中的中和效价分别为1:100 - 1: 3200和（0.1 - 8）×10-4。
成功纯化了GNLY融合蛋白，制备了抗人GNLY的单克隆抗体，为其实验室及临床研究奠定了一定基础。
