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家蚕丝腺具有高效合成与分泌蚕丝蛋白质的能力,探究利用转基因方法开发家蚕丝腺为生物反应器生产高附加值外源蛋白质的技术途径.将Ⅰ型类人胶原蛋白基因(HCool-Ⅰ)和胶原蛋白羟基化修饰所必需的脯氨酰4-羟化酶α-亚基基因(BmP4Hα)通过piggyBac转座子及显微注射方法,同时转入家蚕早期胚胎,在丝素轻链基因fib-L启动子驱动下转入的2个外源基因在家蚕幼虫后部丝腺获得表达.利用反向PCR和生物信息学等方法鉴定、分析转基因家蚕基因组中有19个外源pig-gyBac的整合位点,分别位于基因的外显子区域、内含子区域和基因间区,可定位至家蚕的15条染色体上.实时荧光定量PCR检测不同整合位点的转基因家蚕品系幼虫后部丝腺中HCool-Ⅰ基因mRNA转录量存在板显著差异,转录量最高和最低的品系间相差23倍左右;转基因家蚕后部丝腺中BmP4Hα基因mRNA的转录量比野生型家蚕高出2万多倍.利用SDS-PAGE检测转基因家蚕G3代5龄5d幼虫的后部丝腺总蛋白质在55 kD和64 kD处分别存在特异性蛋白条带,表明BmP4Hα和HCool-Ⅰ蛋白已在G3代转基因家蚕丝腺中获得表达.“,”Bombyx mori silkgland has powerful ability to synthesize and secrete silk protein.In present study,we explored the technological approaches to produce high value exogenous proteins using Bombyx mori silkgland bioreactor developed by transgenic method.Using piggyBac transposon,type Ⅰ huaman collagen-like gene (HCool-Ⅰ) and prolyl 4-hydroxylase α-subunit gene (BmP4Hα) which is required for hydroxylation modification of collagen were co-transferred into the early embryos of silkworm after microinjection.And these two exogenous genes were expressed in the posterior silkgland under the control of light-chain gene (fib-L) promoter.By using reverse PCR combined with bioinformatics analysis,19 exogenous piggyBac integration sites in the genome of transgenic silkworm were identified.These integration sites located in exon,intron and intergenic region,respectively,and mapped to 15 chromosomes.Real-time fluorescent quantitative PCR indicated that there was extremely significant difference in mRNA transcriptional levels of HCool-Ⅰ among various transgenic silkworm strains with different integration sites.The maximum level was 23 times to the minimum.The mRNA transcriptional level of BmP4Hα in posterior silkgland of transgenic silkworm was twenty-thousand times to that of the wild-type silkworm.SDS-PAGE analysis of total proteins in posterior silkgland of day 5 transgenic silkworm larvae of the 5th instar of G3 generation showed that there were specific protein bands of 55 kD and 64 kD,demonstrating that BmP4Hα and HCool-I proteins were expressed successfully in silkgland of the transgenic silkworm of G3 generation.