真核表达人生长激素促进家兔生长及免疫原性分析

陈金武 齐璐璐 段莹莹

摘要 [目的]获得稳定表达重组人生长激素的中华仓鼠卵巢细胞株，并分析目的蛋白体内促动物生长活性及免疫原性。[方法]PCR扩增获得rhGH基因，构建重组表达质粒rhGH1 pOptiVEC，并进行双酶切及测序鉴定。脂质体法转染CHO/DG44 dhfr 细胞，利用SDS PAGE电泳及Western blot检测目的蛋白，并用氨甲喋呤（MTX）加压筛选阳性克隆。采用Co2+亲和层析法纯化rhGH。促去垂体家兔生长法检测rhGH体内活性，同时采用ELISA双抗夹心法检测重组蛋白诱生抗体滴度，分析其免疫原性。[结果]PCR扩增得到696 bp的rhGH序列，双酶切及测序结果显示构建的rhGH1 pOptiVEC质粒正确。转染细胞后，MTX加压使得蛋白表达量明显提高。纯化的rhGH能够促进去垂体家兔体重增加。诱生抗体滴度分析结果显示该蛋白具有与市售商品化蛋白相似的免疫原性。[结论]真核表达rhGH蛋白能够促进动物生长，可用于相关药物研究。

关键词 重组人生长激素；真核表达； 促生长活性；免疫原性

中图分类号：R335；Q291 文献标识码：A 文章编号：2095-3305（2018）03-071-04

DOI： 10.19383/j.cnki.nyzhyj.2018.03.029

Abstract [Objective]To construct the stable expressing vector rhGH1 pOptiVEC for recombinant human growth hormone in CHO/DG44 dhfr cells and identified the growth promoting activity and immunogenicity. [Methods]rhGH gene was amplified by PCR. The PCR product and pOptiVEC vector were digested by double enzymes. Then the two digested products were jointed together with T4 ligase. Then the plasmid was transfected to CHO/DG44 dhfr cells and the positive clones were further screened by different concentration of MTX. The target protein was purified by Co2+ affinity chromatography and analyzed by SDS PAGE electrophoresis & western blot. The body weight gain tests of hypophysectomized rabbits were carried out for potency determination of rhGH in vitro. Rabbits were immunized with rhGH and the serum antibody was also determined by ELISA.[Results]The rhGH1 pOptiVEC vector was constructed correctly， which was proved by double enzymes digestion and DNA sequencing. The positive CHO/DG44 dhfr clones could stably express rhGH with 1000 nmol/L MTX and the molecular mass of the target protein was about 23 kDa. rhGH could be purified to a high concentration（90%）by Co2+ affinity chromatography. It also could promote hypophysectomized rabbits to grow up. The antibody induced by rhGH was similar to that induced by the commercially available drug.[Conclusions]The CHO/DG44 dhfr cells which can highly express rhGH had been successfully constructed. The rhGH protein with the similar growth promoting activity and immunogenicity to the commercially available drug had been also obtained.

Key words Recombinant human growth hormone（rhGH）；Eukaryotic expression；Growth promoting activity；Immunogenicity

人生长激素（human growth hormone，hGH）是一个由191个氨基酸残基组成的非糖基化蛋白质激素[1]，分子量为22 KDa。该蛋白由脑垂体前叶嗜酸性细胞分泌，蛋白质分泌量一般在青春期达到最大值[2-3]。目前人生长激素主要用于治疗儿童的生长障礙和成人生长激素缺乏症（GHD），且具有良好的效果。同时，生长激素也被批准用于特纳综合症、慢性肾功能不全、儿童特发性身材矮小、烧伤烫伤手术后的恢复以及脂肪积累相关的成人脂肪代谢障碍等[4]。早期，hGH主要来自于人类尸体脑垂体，产量非常低，因此科学家尝试多种途径人工合成GH。……