摘要
蜕皮抑制激素(Moltinhibitinghormone,MIH)属于甲壳动物高血糖激素家族神经肽,对甲壳类的蜕皮起抑制作用。本研究用DNA重组技术将中华绒螯蟹(Eriocheirjaponicasinensis)的蜕皮抑制激素1(ErsMIH1)成熟肽的cDNA序列亚克隆至原核表达载体pET28a(+)中,并在大肠杆菌BL21(DE3)中进行高效表达。SDSPAGE检测结果显示,融合蛋白pET-MIH1的Mr约为12kD,与理论值相符。融合蛋白的表达量约占菌体总蛋白的15%,表达产物以包涵体形式存在。对包涵体进行变性、复性及纯化处理,并以8mol/L尿素溶解的包涵体作为免疫原免疫BALB/c小鼠制备多克隆抗体。ELISA和Westernblot的结果表明制备的抗体效价高。
Molting in crustaceans is inhibited by the molt-inhibiting hormone （MIH）, a neuropeptide member of the CHH family. The eDNA encoding the molt-inhibiting hormone 1 （Ers-MIHI） of the mitten crab Eriocheir japonica sinensis was inserted into pET28a （ ＋ ） bacterial expression vector by using recombinant DNA techniques and the fusion protein was highly expressed in inclusion bodies in E.coli BL21 （DE3）. SDS-PAGE analysis revealed that the fusion protein pET-MIHI （Mr 12 kD） was successfully expressed and the resulting MIH1 fused protein was 15 percent of total proteins. By denaturation, renaturation and washing, the inclusion body was purified. Inclusion bodies dissolved in the solution of 8 mol/L urea were used as immunogen to prepare the polyclonal antibody in BALB/c mouse. The quality of the antibody was identified by ELISA and Western blot. The results indicated that polyclonal antiMIH1 antibody with high titer and specificity has been prepared [ Acta Zoologica Sinica 52 （1）： 209- 214, 2006].
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2006年第1期209-214,共6页
ACTA ZOOLOGICA SINICA
基金
国家自然科学基金重点项目资助(No.30130040)~~
关键词
中华绒螯蟹
蜕皮抑制激素
复性
多克隆抗体
基因表达
Eriocheir japonica sinensis, Molt-inhibiting hormone, Renaturation, Polyclonal antibody
