真核表达人生长激素促进家兔生长及免疫原性分析

Analysis on growth promoting activity and immunogenicity of eukaryotic expression recombinant human growth hormone

[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rh GH基因,构建重组表达质粒rh GH1-p Opti VEC,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rh GH。促去垂体家兔生长法检测rh GH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rh GH序列,双酶切及测序结果显示构建的rh GH1-p Opti VEC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rh GH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rh GH蛋白能够促进动物生长,可用于相关药物研究。

[Objective]To construct the stable expressing vector rh GH1-p Opti VEC for recombinant human growth hormone in CHO/DG44 dhfr-cells and identified the growth promoting activity and immunogenicity. [Methods]rh GH gene was amplified by PCR. The PCR product and p Opti VEC 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 rh GH in vitro. Rabbits were immunized with rh GH and the serum antibody was also determined by ELISA. [Results]The rh GH1-p Opti VEC vector was constructed correctly, which was proved by double enzymes digestion and DNA sequencing. The positive CHO/DG44 dhfr-clones could stably express rh GH with 1000 nmol/L MTX and the molecular mass of the target protein was about 23 k Da. rh GH 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 rh GH was similar to that induced by the commercially available drug. [Conclusions]The CHO/DG44 dhfr-cells which can highly express rh GH had been successfully constructed. The rh GH protein with the similar growth promoting activity and immunogenicity to the commercially available drug had been also obtained.