家蚕表达人表皮生长因子gp67信号肽融合蛋白及生物活性的研究

Expression of Fusion Protein of hEGF and gp67 Signal Peptide in Larvae of Bombyx mori

摘要:

人表皮生长因子(hEGF), 一种由53个氨基酸残基组成的单链多肽, 具有广阔的应用前景。本文主要探讨家蚕表达人表皮生长因子gp67信号肽融合蛋白的生物活性。采用家蚕杆状病毒表达系统来表达该信号肽融合蛋白。构建了重组质粒pBacPAKS-hEGF, 将该重组质粒与线性化病毒Bm-BacPAK6 DNA共转染家蚕细胞, 筛选获得重组病毒vBacPAK-SEGF, 用vBacPAK-SEGF感染家蚕BmN细胞和五龄蚕, Western blot检测表明在家蚕细胞、五岭幼虫的血淋巴和蛹中均有约12 kD的目的蛋白表达。ELISA检测发现在家蚕细胞中的表达量为23 mg/ 106细胞, 五龄幼虫中的表达量可达到82 mg/mL血淋巴。利用小鼠成纤维细胞Balb/c3T3分析家蚕表达的hEGF信号肽融合蛋白的生物活性, 结果表明表达产物能显著促进Balb/c3T3细胞的增值。另外, 研究还发现hEGF信号肽融合蛋白可使新生ICR小鼠体重增 加, 睁眼和萌齿时间提前。本研究为进一步开发利用家蚕表达的hEGF提供理论基础。

Abstract:

Bombyx mori baculovirus expression vector system (BEVS) was adopted to express S-hEGF. The reconstructed plasmids of pBacPAKS-hEGF and an engineering modified BmNPV Bm-BacPAK6 DNA were used to co-transfect BmN cells in order to obtain recombinant virus. The recombinant virus (named vBacPAKS-hEGF) was then used to infect BmN cultured cells, the fifth instars larvae and pupa. The aimed 12 kD protein expressed in BmN cultured cells and the fifth instars larvae was identified with Western blot. ELISA results showed that the amount of expression protein reached 23 mg/ 106 in BmN cells and 82 mg/mL in larvae. The biological activity was determined by epidermal growth factor dependent Balb/c3T3 cell line. Both cellular extracts and haemolymph of silkworm larvae infected with the recombinant virus accelerated proliferation of Balb/c3T3 cells. In addition, animal experiment results revealed that S-hEGF fusion protein promoted newborn ICR mice weight increasing, incisor eruption and eyelid opening.