利用柞蚕核型多角体病毒(ApNPV)表达载体系统在柞蚕蛹中表达人生长激素  

Expression of Human Growth Hormone inAntheraea pernyi Pupa Using Antheraea pernyi Nucleopolyhedrovirus Expression Vector System

作　　者：叶博[1] 王林美[1] 赵振军[1] 岳冬梅[1] 张波[1] 李树英[1] 范琦[1] 

机构地区：[1]辽宁省农业科学院大连生物技术研究所,辽宁省野蚕重点实验室,辽宁大连116024

出　　处：《蚕业科学》2014年第2期277-283,共7页ACTA SERICOLOGICA SINICA

基　　金：辽宁省科技厅农业攻关计划项目(No.2011208000)

摘　　要：为探究柞蚕核型多角体病毒（ApNPV）表达载体一柞蚕蛹表达系统用于表达哺乳动物活性蛋白的可行性，利用该表达系统表达医用蛋白人生长激素（hGH）。构建插入hgh基因的转移表达载体pApM748BE／hgh，将该转移表达载体质粒DNA与病毒ApNPV-△ph／Aefp／hgh’基因组DNA共转染樗蚕（Philosamiacynthiam）培养细胞Pc一01，用末端稀释法筛选获得重组病毒ApNPV-△ph／Aefp／hgh。采用Westernblotting和ELISA方法，检测到柞蚕蛹感染重组病毒ApNPV—△ph／-△ph／Aefp／hgh／hgh’后第7天体液中就有hGH明显表达；感染后第13～14天蛹体液中的hGH含量保持较高水平，重组hGH的最高表达量可达251．18肛g／mL；感染后第15～20天仍可以检测到hGH的表达，但表达产物出现降解现象。用活化的人红白血病细胞K562检测利用ApNPV表达载体系统在柞蚕蛹表达的重组hGH的体外活性，结果显示其对K562细胞的增殖具有明显的促进作用。To discuss the possibility of using Antheraea pernyi nucleopolyhedrovirus （ApNPV）-Antheraea pernyi pupa expression system to express active proteins in mamals, human growth hormone （hGH） for medical use was expressed using this expression system. The transfer vector, pApM748BE/hgh, was constructed and co-transfected into Philosamia cynthiam cells （Pc-01） with the genomic DNA of ApNPV-△ph/egfp. The recombinant virus, ApNPV-△ph/△egfp/hgh＋, was obtained by modified plaque purification. Western blotting and ELISA results showed that hGH could be detected in pupal haemolymph from 7 dpi （days post infection） of recombinant virus ApNPV-Aph/Aegfp/hgh＋, and maintained high expression level at 3 to 14 dpi, during which the highest expression level was up to 251.18μg/mL, rhGH expression was detectable from 15 to 20 dpi, but the expressed product started to degrade. The recombinant hGH expressed inAntheraea pernyi pupae using ApNPV expression vector system could obviously stimulate the proliferation of K562 human erythroleukemia cells, as indicated by in vitro ac- tivity assay using the activated K562 human erythroleuke- mia cells.

关 键 词：柞蚕核型多角体病毒表达载体系统 柞蚕蛹 人生长激素 体外活性 

分 类 号：S884.5[农业科学—特种经济动物饲养]