绵羊MSTN多克隆抗体的制备及细胞毒性分析

Production of Polyclonal Anti-Myostatin Antibody of Ovine and the Analysis of Cytotoxicity

目的:研究绵羊肌肉生长抑制素(Myostatin,MSTN)多克隆抗体的安全性。方法:将绵羊MSTN成熟肽基因克隆至原核表达载体pET-32a(+),转化至BL21(DE3)中;利用Ni-NTA亲和层析法纯化目的蛋白,免疫新西兰兔制备多克隆抗体,West-ern blotting鉴定其免疫原性;ELISA检测抗体效价;通过检测乳酸脱氢酶(lactic dehydrogenase,LDH)的水平研究其细胞毒性。结果:成功获得了330 bp的绵羊MSTN成熟肽基因,表达蛋白分子量约为33 kDa,纯度达90%以上;Western blot检测表明MSTN蛋白具有免疫原活性;ELISA检测证实制备的抗体可与抗原蛋白特异性结合,抗体效价为l:25 600,细胞毒性试验表明高浓度的多克隆抗体(200μg/mL)对肌肉细胞无毒害作用。结论:制备的绵羊MSTN多克隆抗体具有生物学活性,对肌肉细胞无细胞毒性,为促生长效果验证提供了安全保障。

Objective ：The security of ovine myostatin polyclonal antibody was studied. Method：The ovine myostatin mature peptide frag- ment was subcloned into the pET-32a （ ＋ ）, and transformed into E. coli BI21; The recombinant fusion protein was purified by Ni - NTA affinity Chromatography and injected into New zealand white rabbit to produce polyclonal anti - myostatin antibody, Western blottingidentified the immunogenicity of the expressed protein ; Titer detected by ELISA ; The cytotoxicity was analyzed by detecting the level of LDH. Result：Its successful to clone a MSTN sequence of 330bp, and express recombinant protein MSTN of 33kDa induced in E. coli, the purity highed than; Western blot demonstrated the myostain protein have immunogen activity; The antibody showed high affinity to the re- combinant myostatin detected by ELISA, and antibody titer for 1：25 600; The cytonoxicity tests showed that a high concentration of poly- clonal antibody of no- toxic effects on muscle cells. Conclusion：This research showed that ovine myostatin polyclonal antibody have bio- logical activity and non - toxic affect on the muscle cells, so provide security for the next verification growth - promoting effect.