家蚕丝腺生物反应器表达hGM-CSF转基因载体的构建及应用研究
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摘要
家蚕(Bombyx mori),又名桑蚕,属鳞翅目家蚕蛾科,是迄今为止数百万种昆虫中仅有的被驯化并大规模人工饲养的经济昆虫。其幼虫具有一对发达的绢丝腺,能够大量合成分泌丝蛋白。近几年,以转基因家蚕表达外源蛋白的技术体系正被逐步建立。但现有的技术体系存在外源基因表达量不高,筛选工作量大等问题。
本研究以丝蛋白基因启动子构建新的基于piggyBac转座子的家蚕转基因载体。克隆丝素蛋白重链基因(fibroin heavy chain,fib-H)、轻链基因(fibroin light chain,fib-L)启动子,并以生物信息学手段分析启动子元件;构建fib-H、fib-L启动子控制红色荧光蛋白(Discosoma red fluorescent protein,DsRed/drFP583)报告基因的瞬时表达载体,进行家蚕体内及BmN培养细胞中的瞬时表达试验;构建分别以fib-H、fib-L启动子控制外源基因的转基因载体;以piggyBac转座子介导进行家蚕培养细胞的转基因研究;以构建完成的转基因载体初步进行家蚕转基因研究。
研究结果获得了fib-H启动子(EF540776,489 bp)、fib-L启动子(EF540777,606 bp),fib-L来源的polyA加尾信号序列(EF216676,289 bp),fib-L第一内含子来源的增强子序列(enhancer)(DQ679478,375 bp),新霉素抗性基因(neomycin resistance gene,neo~r)等元件;瞬时表达证明克隆的启动子在家蚕后部丝腺组织具有特异性活性,同时发现其在家蚕卵巢来源的BmN培养细胞中亦具有一定的活性;以克隆的元件构建二种新的转基因载体,除了已有的绿色荧光蛋白(green fluorescent protein,GFP)报告基因外,载体中包括分别以fib-n、fib-L启动子控制外源功能基因hGM-CSF(humangranulocyte-macrophage colony stimulating factor,粒/巨噬细胞集落刺激因子)并带有polyA加尾信号的表达元件、促进转录活性的增强子元件、可与GFP进行双重筛选的neo~r基因表达元件;以带有neo~r基因表达元件和GFP报告基因的piggyBac转座子载体转化家蚕BmN细胞,以终浓度800μg/mL的G418(Geneticin)筛选3个月,获得稳定转化细胞,呈现绿色荧光细胞数约75%,PCR鉴定证实细胞基因组DNA中外源基因的存在;构建的转基因载体初步进行家蚕转基因试验,幼虫2龄3 d观察到3条蚕体表具有绿色荧光斑点,蛹期观察到3个表现明显绿色荧光的个体。
本研究构建的转基因载体可促进转基因家蚕生物反应器生产外源蛋白的深入研究。
The mulberry silkworm Bombyx mori(Lepidoptera:Bombycidae) is the tamed member of the millions of insects and reared economically in large scale.The larva of silkworm has a pair of highly developed silk glands with the ability to synthesize fibroin.Recently,the technical system has been gradually constructed to express foreign proteins using transgenic silkworms.However,there are some drawbacks in current technical systems such as lower expression level of foreign proteins and heavy selecting jobs.
In the present research,a transgenic vector based on piggyBac transposon for silkworm was constructed by using fibroin promoters.The promoters of fibroin heavy chain(fib-H) and fibroin light chain(fib-L) were cloned and their motifs were analyzed through bioinformatics.Transient expression vectors using DsRed as reporter gene under the control of fib-H and fib-L promoters were constructed and assayed in the body of silkworm and Bran cells.Transgenic vectors used for expressing foreign proteins under the control of fib-H and fib-L promoters were constructed respectively and used in the transgenic research of BmN cells and silkworms.
Promoters of fib-H(EF540776,489 bp),fib-L(EF540777,606 bp),polyA signal sequence(EF216676, 289 bp),enhancer(DQ679478,375 bp) from intron-1 of fib-L gene,and neomycin resistance gene(neo~r) were cloned for the construction of transgenic vectors.The second structures of fib-H and fib-L were analyzed. Transient expression showed that both of the promoters fib-H,fib-L had specific inductive activities in the posterior silk gland,and also a few of activities in BmN cells.
Two transgenic vectors were constructed by respectively combining fib-H and fib-L promoters with hGM-CSF,the polyA signal sequence,enhancer,neo~r and the GFP reporter gene already included in the original vector.Bran cells were transfected by piggyBac transposon with neo~r and GFP double selection elements.Through screen by G418 at the final concentration of 800μg/mL for 3 months,a stable transformation cell strain was obtained.Approximate 75%of the BmN cells were observed emitting green fluorescence.Foreign genes were proved existing in the genome of the cells by PCR.The two constructed transgenic vectors were used to conduct exploration of transgenic experiment on silkworm.The green fluorescence spots were observed in 3 transgenic larvae at 3 day 2~(nd) instar silkworm,and 3 pupa.with green fluorescence was also observed.
The two transgenic vectors constructed in this research may lead deep research on the expression of foreign proteins by transgenic silkworm bioreactor.
本研究以丝蛋白基因启动子构建新的基于piggyBac转座子的家蚕转基因载体。克隆丝素蛋白重链基因(fibroin heavy chain,fib-H)、轻链基因(fibroin light chain,fib-L)启动子,并以生物信息学手段分析启动子元件;构建fib-H、fib-L启动子控制红色荧光蛋白(Discosoma red fluorescent protein,DsRed/drFP583)报告基因的瞬时表达载体,进行家蚕体内及BmN培养细胞中的瞬时表达试验;构建分别以fib-H、fib-L启动子控制外源基因的转基因载体;以piggyBac转座子介导进行家蚕培养细胞的转基因研究;以构建完成的转基因载体初步进行家蚕转基因研究。
研究结果获得了fib-H启动子(EF540776,489 bp)、fib-L启动子(EF540777,606 bp),fib-L来源的polyA加尾信号序列(EF216676,289 bp),fib-L第一内含子来源的增强子序列(enhancer)(DQ679478,375 bp),新霉素抗性基因(neomycin resistance gene,neo~r)等元件;瞬时表达证明克隆的启动子在家蚕后部丝腺组织具有特异性活性,同时发现其在家蚕卵巢来源的BmN培养细胞中亦具有一定的活性;以克隆的元件构建二种新的转基因载体,除了已有的绿色荧光蛋白(green fluorescent protein,GFP)报告基因外,载体中包括分别以fib-n、fib-L启动子控制外源功能基因hGM-CSF(humangranulocyte-macrophage colony stimulating factor,粒/巨噬细胞集落刺激因子)并带有polyA加尾信号的表达元件、促进转录活性的增强子元件、可与GFP进行双重筛选的neo~r基因表达元件;以带有neo~r基因表达元件和GFP报告基因的piggyBac转座子载体转化家蚕BmN细胞,以终浓度800μg/mL的G418(Geneticin)筛选3个月,获得稳定转化细胞,呈现绿色荧光细胞数约75%,PCR鉴定证实细胞基因组DNA中外源基因的存在;构建的转基因载体初步进行家蚕转基因试验,幼虫2龄3 d观察到3条蚕体表具有绿色荧光斑点,蛹期观察到3个表现明显绿色荧光的个体。
本研究构建的转基因载体可促进转基因家蚕生物反应器生产外源蛋白的深入研究。
The mulberry silkworm Bombyx mori(Lepidoptera:Bombycidae) is the tamed member of the millions of insects and reared economically in large scale.The larva of silkworm has a pair of highly developed silk glands with the ability to synthesize fibroin.Recently,the technical system has been gradually constructed to express foreign proteins using transgenic silkworms.However,there are some drawbacks in current technical systems such as lower expression level of foreign proteins and heavy selecting jobs.
In the present research,a transgenic vector based on piggyBac transposon for silkworm was constructed by using fibroin promoters.The promoters of fibroin heavy chain(fib-H) and fibroin light chain(fib-L) were cloned and their motifs were analyzed through bioinformatics.Transient expression vectors using DsRed as reporter gene under the control of fib-H and fib-L promoters were constructed and assayed in the body of silkworm and Bran cells.Transgenic vectors used for expressing foreign proteins under the control of fib-H and fib-L promoters were constructed respectively and used in the transgenic research of BmN cells and silkworms.
Promoters of fib-H(EF540776,489 bp),fib-L(EF540777,606 bp),polyA signal sequence(EF216676, 289 bp),enhancer(DQ679478,375 bp) from intron-1 of fib-L gene,and neomycin resistance gene(neo~r) were cloned for the construction of transgenic vectors.The second structures of fib-H and fib-L were analyzed. Transient expression showed that both of the promoters fib-H,fib-L had specific inductive activities in the posterior silk gland,and also a few of activities in BmN cells.
Two transgenic vectors were constructed by respectively combining fib-H and fib-L promoters with hGM-CSF,the polyA signal sequence,enhancer,neo~r and the GFP reporter gene already included in the original vector.Bran cells were transfected by piggyBac transposon with neo~r and GFP double selection elements.Through screen by G418 at the final concentration of 800μg/mL for 3 months,a stable transformation cell strain was obtained.Approximate 75%of the BmN cells were observed emitting green fluorescence.Foreign genes were proved existing in the genome of the cells by PCR.The two constructed transgenic vectors were used to conduct exploration of transgenic experiment on silkworm.The green fluorescence spots were observed in 3 transgenic larvae at 3 day 2~(nd) instar silkworm,and 3 pupa.with green fluorescence was also observed.
The two transgenic vectors constructed in this research may lead deep research on the expression of foreign proteins by transgenic silkworm bioreactor.
引文
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