摘要
AIM： To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria rneningitids and to compare their immunogenic effect. METHODS： G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21（DE3）. After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done, Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. RESULTS： G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84^th day after the first immunization, the titer of antibody against cross-linked protein reached 51 200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. CONCLUSION： The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.
瞄准：从奈瑟氏菌属 meningitids 基于 P64K 蛋白质构造二种 anti-gastrin 免疫情报并且比较他们的产生免疫性的效果。方法：G17P64K 基因被克隆并且绑扎进 pET28a 原生质标志，然后转变了成 BL21 (DE3 ) 。在磅媒介和 IPTG 正式就职的接种以后， recombinant 蛋白质 solubly 在高水平被表示。G17P64K 熔化蛋白质的纯化类似于 P64K 的。纯化由 30% 组成的起始的步浸透了硫酸铵降水被做。另外的好优化包括了 phenyl-sepharose， G200 Sephadex 胶化过滤和 Q-sepharose 阴离子 exchanger 层析。高度净化的蛋白质在 N 终端氨基酸残余被获得并且定序。多肽被 MBS 方法然后兔子 anti-gastrin 被 Fmoc 稳固的阶段化学法和 cross-linked 综合到搬运人蛋白质 P64K 和 DT 异种 17 抗体被与 cross-linked 使兔子免疫准备并且熔化了蛋白质。效价和抗体的活动在试管内被估计。结果：G17P64K 基因和 recombinant 细菌被获得。在四步纯化以后，超过 90% 有纯净的蛋白质样品被完成。在 84 (th ) 在第一免疫以后的白天，对 cross-linked 蛋白质的抗体的效价到达了 51,200。抗体在试管内的评估表明了那它在肿瘤房间 SW480 的生长上举办了一项高禁止的活动。结论：P64K 多肽 cross-linked 免疫情报使兔子免疫并且对促胃液素完成了更高的效价抗体 17 比 G17P64K 熔化蛋白质免疫情报，它能禁止肿瘤细胞 SW480 的生长。
基金
Supported by Grants from National High Technology Research and Development Program, No.2002AA2Z345B and No.2004AA2Z3803 of the Ministry of Science and Technology of China
