Xiong Xiang-Hua, Zhao Hong-Liang, Xue Chong, Zhang Wei, Yang Bing-Fen, Yao Xue-Qin, Liu Zhi-Min
Department of Microbiologic Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.
World J Gastroenterol. 2006 Apr 7;12(13):2040-6. doi: 10.3748/wjg.v12.i13.2040.
To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect.
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.
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.
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.
基于脑膜炎奈瑟菌的P64K蛋白构建两种抗胃泌素免疫原，并比较它们的免疫原性效果。
克隆G17P64K基因并连接到pET28a质粒中，然后转化到BL21(DE3)中。接种LB培养基并经IPTG诱导后，重组蛋白以高水平可溶性表达。G17P64K融合蛋白的纯化方法与P64K相似。首先进行30%饱和硫酸铵沉淀的初步纯化步骤。进一步的精细优化包括苯基琼脂糖、G200葡聚糖凝胶过滤和Q-琼脂糖阴离子交换柱层析。获得了高度纯化的蛋白并对其N端氨基酸残基进行测序。采用Fmoc固相化学方法合成多肽，并通过MBS方法将其与载体蛋白P64K和DT突变体交联，然后用交联融合蛋白免疫兔制备抗胃泌素17抗体。评估抗体的效价和体外活性。
获得了G17P64K基因和重组菌。经过四步纯化后，得到了纯度高于90%的蛋白样品。首次免疫后第84天，抗交联蛋白抗体的效价达到51,200。抗体的体外评估表明，它对肿瘤细胞SW480的生长具有高抑制活性。
P64K-多肽交联免疫原免疫兔后，获得了比G17P64K融合蛋白免疫原更高效价的抗胃泌素17抗体，该抗体可抑制肿瘤细胞SW480的生长。
