蓖麻蚕蛹生物反应器生产重组人表皮生长因子的研究  

Research for the Production of Recombinant Human Epidermal Growth Factor Using Samia Cynthia Ricini Pupae Bioreactor

作　　者：伍玉婷[1] 黄元姣[2] 雷丹青[2] 吴勇浒[1] 李牡艳[2] 小林淳[3] 王先裕[4] 

机构地区：[1]广西医科大学研究生学院,南宁530021 [2]广西医科大学医学科学实验中心,南宁530021 [3]日本山口大学农学部 [4]广西大学农学院,南宁530004

出　　处：《生物医学工程学杂志》2013年第1期136-140,共5页Journal of Biomedical Engineering

基　　金：广西科学研究与技术开发计划项目资助(10100019-20)

摘　　要：重组人表皮生长因子(rhEGF)已经广泛应用于医疗和化妆品。以杆状病毒AnpeNPV为基因表达载体,昆虫为生物反应器而建立的蛋白表达系统已获得成功。本文以人表皮生长因子(hEGF)基因替代柞蚕AnpeNPV中的核多角体基因而获得的AnpehEGF病毒为载体、蓖麻蚕蛹为生物反应器表达的rhEGF蛋白质,采用PCR、West-ern blot和ELISA等实验方法对其进行检测,硫酸铵沉淀和Ni-NTA Agrose亲和层析法对其进行分离纯化。实验结果显示,无论是在基因或是蛋白水平上都可检测到rhEGF的表达。AnpehEGF感染蚕蛹后第6d开始检测到rhEGF的表达量快速上升,第12d达到高峰,第3、6、9、12d的表达量分别为19.77、24.90、618.59、1 952.46ng/g,而到了病毒感染后期(第15d)出现了蛋白降解现象。表达的rhEGF通过分离纯化获得了较纯的产品。结果表明蓖麻蚕蛹作为生物反应器生产外源蛋白rhEGF是可行的,说明利用AnpeNPV和蓖麻蚕蛹可开发更加低成本而又高效的rhEGF生产新途径。The protein production system using a baculovirus Antheraea pernyi nucleopolyhedrovirus （AnpeNPV） as a gene expression vector and its host insect as a natural bioreactor was successful established and its excellent per- formance in the protein production has been demonstrated. In this paper, the system is used to produce recombinant human epidermal growth factor （rhEGF）, which have been widely used in medical and cosmetic treatment. A recom- binant AnpehEGF virus has been constructed by replacing the viral polyhedrin gene with the rhEGF gene, and then injected it to Samia cynthia ricini pupae. Amplification and expression of rhEGF gene in the pupae was clearly de- tected by PCR, Western blot and ELISA analyses. These analyses have also revealed that rhEGF in the pupae was significantly increased at 6 days post-infection, and reached maximum level at the 12th day. The concentrations of rhEGF were 19.77, 24.90, 618.59 and 1 952.46 ng/g pupae at 3, 6, 9 and 12 days post-infection, respectively. However, the rhEGF concentration reduced at later stage （days 15）. The rhEGF in the pupae could be purified using ammonium sulfate precipitation and Ni-NTA agrose affinity chromatography. Results demonstrate that Sarnia cyn- thia ricini pupae can be used as a bioreactor to produce rhEGF and, if successfully improved, will be a novel method of rhEGF production with lower cost and more efficient.

关 键 词：蓖麻蚕蛹 生物反应器 重组人表皮生长因子 

分 类 号：Q78[生物学—分子生物学] Q74