基因打靶转基因桑蚕载体的构建及杆状病毒几丁质酶基因（ChiA）和半胱氨酸蛋白酶基因（CP）失活表达GIL－3的研究
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摘要
随着经济和科技的发展，以桑蚕作为生物反应器生产有用蛋白为古老的蚕丝业注入了新的活力。桑蚕生物反映器主要有两种形式：杆状病毒表达载体系统(Baculovirus Expression Vector System，BEVS)和转基因桑蚕(transgenicsilkworm)。
我们克隆了家蚕核型多角体病毒苏州株的IE-1启动子的全序列和桑蚕丝素蛋白基因轻链第五外显子到第七外显子之间的1.2kb和0.5kb的片段作为侧翼序列，将置于IE-1启动子之下的绿色荧光蛋白基因(GFP)插入其中，期待经过基因打靶可以得到单拷贝的转基因桑蚕。
本文用含多角体蛋白基因的重组杆状病毒转移载体pSKC-P-C与能表达白介素-3(IL-3)的重组杆状病毒BmNGIL-3在桑蚕细胞中进行共转染，通过同源重组、经两轮空斑纯化，获得了几丁质酶基因(ChiA)和半胱氨酸蛋白酶基因(CP)失活、可形成多角体的、并表达IL-3的重组杆状病毒NdBmNGIL-3。NdBmNGIL-3感染细胞的存活时间比普通病毒长，推测ChiA、CP二基因的失活可延长外源基因的表达水平。
With the growth of economic and technology, it's possible to use the silkworm as a bio-reactor for production of heterologous protein. There are two main ways to produce interesting protein by silkwrom, the baculovirus expression vector system (BEVS) and transgenic silkworm.
We coloned BmNPVsu IE-1 promoter, 1.2kb and 0.5kb gene of fibroin light chain from the 5th exon to the 7th exon to construct the transgenic silkworm vector. By means of insert report gene GFP which is downstream of IE-1 promoter in the fibroin gene we have coloned, it's possible to get the single copy gene of trasgenic silkworm which will spin silk and express the gene continually.
We construct the vector pSKC-P-C which has Polh gene between ChiA and CP. The vector was infected BmN cells with BmNGIL-3 so that the ChiA and CP are out of function. The recombinant virus NdBmNGIL-3 we gained has showed that the expression time of GIL-3 was prolonged and the polh and GIL-3 was detected in BmN cells, accordingly, recombinant virus could be infected silkworm by eaten.
我们克隆了家蚕核型多角体病毒苏州株的IE-1启动子的全序列和桑蚕丝素蛋白基因轻链第五外显子到第七外显子之间的1.2kb和0.5kb的片段作为侧翼序列，将置于IE-1启动子之下的绿色荧光蛋白基因(GFP)插入其中，期待经过基因打靶可以得到单拷贝的转基因桑蚕。
本文用含多角体蛋白基因的重组杆状病毒转移载体pSKC-P-C与能表达白介素-3(IL-3)的重组杆状病毒BmNGIL-3在桑蚕细胞中进行共转染，通过同源重组、经两轮空斑纯化，获得了几丁质酶基因(ChiA)和半胱氨酸蛋白酶基因(CP)失活、可形成多角体的、并表达IL-3的重组杆状病毒NdBmNGIL-3。NdBmNGIL-3感染细胞的存活时间比普通病毒长，推测ChiA、CP二基因的失活可延长外源基因的表达水平。
With the growth of economic and technology, it's possible to use the silkworm as a bio-reactor for production of heterologous protein. There are two main ways to produce interesting protein by silkwrom, the baculovirus expression vector system (BEVS) and transgenic silkworm.
We coloned BmNPVsu IE-1 promoter, 1.2kb and 0.5kb gene of fibroin light chain from the 5th exon to the 7th exon to construct the transgenic silkworm vector. By means of insert report gene GFP which is downstream of IE-1 promoter in the fibroin gene we have coloned, it's possible to get the single copy gene of trasgenic silkworm which will spin silk and express the gene continually.
We construct the vector pSKC-P-C which has Polh gene between ChiA and CP. The vector was infected BmN cells with BmNGIL-3 so that the ChiA and CP are out of function. The recombinant virus NdBmNGIL-3 we gained has showed that the expression time of GIL-3 was prolonged and the polh and GIL-3 was detected in BmN cells, accordingly, recombinant virus could be infected silkworm by eaten.
引文
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219-229
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