利用Bac-to-Bac杆状病毒表达系统在家蚕中表达人瘦素蛋白

Expression of Human Leptin in Bombyx mori Using Bac-to-Bac Bacu-lovirus Expression System

瘦素(leptin)蛋白由肥胖基因编码,对人体能量代谢等多种生理过程起着重要的调节作用。利用Bac-to-Bac杆状病毒表达系统,将人瘦素蛋白基因(lep)克隆到杆状病毒转移载体pFastBacHTb,并转化E.coli DH10Bac,获得重组杆粒Bacmid-lep,然后转染家蚕BmN细胞,获得重组杆状病毒。用SDS-PAGE和Western blotting方法在感染重组病毒的家蚕BmN细胞检测到大小约22 kD的蛋白条带,与预期的人瘦素蛋白分子质量相符。给家蚕5龄起蚕注射重组病毒液后的4～5 d出现发病症状,RT-PCR检测发病家蚕的血液中有lep基因转录,并通过SDS-PAGE和Western blotting检测到大小为22 kD的人瘦素蛋白的表达。研究结果表明,利用Bac-to-Bac表达系统能够获得含有人瘦素蛋白基因的重组杆状病毒,并且目的蛋白能在家蚕细胞及幼虫体内表达。

Leptin is coded by obese gene.It plays important regulatory roles in energy metabolism and a number of other physiological processes of human.Using the Bac-to-Bac baculovirus expression system,a human lep gene was subcloned into the baculovirus transfer vector pFastBacHTb and was transformed into E.coli DH10Bac to construct recombinant Bacmid-lep.Bacmid-lep was then used to transfect BmN cells to obtain the recombinant baculovirus.SDS-PAGE and Western blotting detected a protein band of about 22 kD in BmN cells infected by the recombinant virus,which was correspondent to the deduced molecular weight of human leptin.After the newly exuviated larvae of the 5th instar were injected with the recombinant virus,the symptoms of infection appeared 4 to 5 d later.RT-PCR detection showed transcription of lep gene in the hemolymph of infected larvae.SDS-PAGE and Western blotting also detected expression of human leptin with 22 kD size in the hemolymph.The above results indicated that the Bac-to-Bac expression system can be use to prepare recombinant baculovirus containing human lep gene and the target protein can be expressed in BmN cells and Bombyx mori larvae.