家蚕丝腺生物反应器表达狂犬病病毒核蛋白

【目的】采用家蚕丝腺生物反应器这一蛋白高效表达系统，尝试建立一种高效、低廉、稳定生产狂犬病病毒核蛋白（RVNP）的技术体系，为生产基因工程疫苗奠定基础。【方法】通过RT-PCR方法克隆获得RVNP序列，采用家蚕丝胶蛋白基因（Ser1）启动子为特异性启动子，将RVNP构建到含有增强型绿色荧光蛋白（EGFP）筛选标记基因的piggyBac转座子表达载体（pBA3EGFP）中，并采用显微注射转基因家蚕技术构建家蚕丝腺生物反应器。【结果】G1代通过EGFP标记的筛选获得转基因家蚕阳性蛾区26个，蛾圈阳性率为59.09%。PCR实验证实RVNP已经整合到家蚕基因组中，Western blot分析表明RVNP蛋白可能附着于丝胶蛋白并随家蚕吐丝行为分泌到蚕茧中。【结论】获得了能够表达外源的RVNP蛋白的转基因家蚕品系，该系统有望成为高效、低廉、稳定地生产狂犬病基因工程疫苗的技术体系之一。

Expression of RVNP in the Silk Gland of Transgenic Silkworm (Bombyx mori)

【Objective】In order to obtain a potent technology which can produce high value RVNP protein and then lay a foundation for the production of vaccines, the silk gland bioreactor was employed as an expression system in this study.【Method】The nucleoprotein gene of rabies virus ERA strain (RVNP) was cloned by RT-PCR, and the RVNP was firstly connected to the downstream of middle silk gland-specific Ser1 promoter, and then inserted into pBA3EGFP, an expression vector which contained an EGFP reporter gene. By using the transgenic method, the silk gland bioreactor of piggyback-mediated transgenic silkworm was obtained. 【Result】Twenty-six broods of transgenic silkworms (G1) were obtained and the GFP-positive percentage of broods was 59.09%. Further PCR identification indicated that RVNP had been integrated into silkworm genome, and Western blot analysis showed that the protein might be attached to the sericin which was secreted into the cocoon with the spinning of transgenic silkworms. 【Conclusion】Transgenic silkworm strain able to express exogenous RVNP protein was obtained. The present system is expected to become a high efficiency, low-cost and stable technical system to produce genetic engineering rabies vaccine.