家蚕丝素重链启动子驱动DsRed的瞬时分泌表达

根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,克隆了家蚕丝素重链基因(Fib-H)启动子及其下游的信号肽序列(FibHS),将DsRed基因与信号肽序列融合构建了分泌型瞬时表达载体;转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed;家蚕注射载体后,可在丝腺腔中检测到红色荧光,表明瞬时表达的DsRed分泌到丝腺腔,推测所克隆的序列具有信号肽的功能。此外,本研究为家蚕丝腺生物反应器分泌表达外源基因的研究奠定了基础。     

家蚕丝蛋白Fhx/P25基因启动子区域的克隆及序列分析

为研究家蚕Fhx/P25基因在时空上的调控机制,通过PCR扩增获得家蚕丝素蛋白Fhx/P25基因的启动子序列并进行克隆和序列分析。进一步构建了由Fhx/P25启动子驱动报告基因DsRed的表达载体pSK-P25-DsRed-PolyA,并通过家蚕BmN细胞进行瞬时表达。结果显示:Fhx/P25基因的启动子序列符合真核生物启动子特点,具有丝腺特异性表达启动子的特征,TATA框的保守序列为TATAA,位于-28—-32处,CAAT基序有3个,其中-110—-117和-90—-87处的2个CAAT基序可能具有活性;二级结构分析显示:Fhx/P25启动子区域具有复杂的茎环结构,这可能与蛋白表达的组织特异性、时间性以及活性有关。基因启动子可以驱动红色荧光基因DsRed在家蚕BmN培养细胞中的瞬时表达。     

家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

家蚕丝蛋白Fhx/P25基因启动子区域的克隆及序列分析

为研究家蚕Fhx/P25基因在时空上的调控机制，通过PCR扩增获得家蚕丝素蛋白Fhx/P25基因的启动子序列并进行克隆和序列分析。进一步构建了由Fhx/P25启动子驱动报告基因DsRed的表达载体pSK-P25-DsRed-PolyA，并通过家蚕BmN细胞进行瞬时表达。结果显示：Fhx/P25基因的启动子序列符合真核生物启动子特点，具有丝腺特异性表达启动子的特征，TATA框的保守序列为TATAA，位于-28—-32处，CAAT基序有3个，其中-110—-117和-90—-87处的2个CAAT基序可能具有活性；二级结构分析显示：Fhx/P25启动子区域具有复杂的茎环结构，这可能与蛋白表达的组织特异性、时间性以及活性有关。基因启动子可以驱动红色荧光基因DsRed在家蚕BmN培养细胞中的瞬时表达。     

家蚕hsp20.4启动子克隆及其驱动表达产物EGT对家蚕蛹体发育的影响

为验证家蚕Bombyx mori热休克蛋白基因hsp20.4启动子的活性以及家蚕核多角体病毒egt的表达产物对家蚕发育的影响, 本实验通过PCR扩增分别得到hsp20.4启动子片段和egt片段。利用hsp20.4的启动子和红色荧光蛋白报告基因DsRed构建重组载体, 在家蚕BmN细胞以及家蚕组织中得到了瞬时表达, 表明所克隆的hsp20.4启动子序列具有热休克蛋白基因的启动子活性。又利用hsp20.4启动子和家蚕核多角体病毒的egt构建重组载体, 通过注射到蚕蛹中进行瞬时表达, 以检测egt表达产物对家蚕发育的影响, 经42℃ 1 h热诱导后, hsp20.4启动子控制的egt表达产物可以延迟家蚕发育。     

家蚕云7A Sericin1基因启动子克隆及活性分析

家蚕Sericin1基因(Ser1)是中部丝腺特异表达基因,其启动子在家蚕生物反应器研究方面发挥着重要作用。为了解云南家蚕品种云7 Ser1基因上游调控序列存在的多态性,进而获得具有驱动活性的Ser1启动子序列,克隆了家蚕Ser1基因启动子并进行序列分析,构建了由该基因启动子驱动红色荧光蛋白基因Ds Red的表达载体p Bac[Ser1p-Ds RedSV40+A3-EGFP],转染家蚕Bm N细胞进行瞬时表达来验证启动子活性。该启动子同已报道Ser1基因上游调控序列比对发现,顺式作用元件区域高度保守,而其他区域存在422 bp的碱基缺失;Ser1基因上游调控序列中存在启动子TATA框和CAAT框分别位于-24~-30处和-112~-115处,其中TATA框的保守序列是TATAAAA;而启动子驱动下的Ds Red基因在Bm N细胞和中部丝腺中成功表达。结果表明云南家蚕品种Ser1上游调控序列存在多态性和驱动活性,为了解不同家蚕品种丝胶合成能力差异和获得高效启动子奠定了基础。     

四种启动子调控RFP报告基因在家蚕细胞(Bm-e-HNU5)内的瞬时表达

以红色荧光蛋白基因（RFP）为报告基因,构建含4种不同启动子的重组表达质粒,用脂质体介导法转染家蚕Bombyx mori细胞（Bm-e-HNU5）,观察家蚕细胞质肌动蛋白4基因启动子（A4）、α微管蛋白基因启动子（α-tub）、蚕丝心蛋白重链基因启动子（Fib）和家蚕核型多角体病毒早期即刻蛋白基因启动子（IE）4种启动子调控RFP报告基因在家蚕细胞内的瞬时表达情况。构建的重组表达质粒pDsRed-α-tub、pDsRed-A4、pDsRed-IE和pDsRed-Fib经双酶切和PCR鉴定正确无误。转染和转录实验结果表明,除了pDsRed-A4外,其他3种重组质粒在Bm-e-HNU5细胞中都得到高转染率,α-tub、IE和Fib可依次增强RFP报告基因在家蚕细胞内的瞬时表达活性。     

家蚕丝蛋白基因启动子调控β－半乳糖苷酶在家蚕细胞系中的瞬时表达

家蚕丝蛋白基因启动子调控β－半乳糖苷酶在家蚕细胞系中的瞬时表达华刚,钱斌,吴祥甫（中国科学院上海生物化学研究所,２０００３１）关键词丝蛋白启动子;β－半乳糖苷酶基因;瞬时表达家蚕丝蛋自基因（Ｗｂ：Ｆｉｂｒｏｉｎｇｅｎｅ）在发育后期幼虫的后部丝腹中主要...     

家蚕五龄幼虫后部丝腺细胞EST的测定和基因表达谱分析

以C108蚕品种五龄第1天和第5天的后部丝腺为材料, 依靠EST分析探讨了有关后部丝腺细胞合成分泌丝素蛋白基因的表达调控问题. 研究结果发现, 测得的9948条EST序列, 经序列拼接分析得到2861个一致性序列, 其中, 重叠的EST群有911个, 单条序列有1950个; 被注释的一致性序列达1335个, 未被注释的一致性序列达1526个; 55.89%, 共5560条与Mita等人发表的家蚕后部丝腺细胞EST没有同源性; 五龄第1天得到的重叠的EST群数和单条序列数都要比五龄第5天的多约1倍; 重叠的EST群组成大小大于50的基因仅占所有一致性序列的0.5%左右, 基因表达的频率开差非常大, 主要表达的基因是与丝素组成和丝素分泌合成有关的基因; 五龄第5天基因表达量与第1天相比, 丝素重链基因高18倍, 丝素轻链基因高9倍, 丝素P25基因高8倍; 经功能注释分析, 被赋予担负细胞组分功能的基因达508个, 担负酶功能的基因达315个. 结果暗示了五龄第1天的基因表达除了作用于丝腺细胞的生长外, 主要是为丝素蛋白合成分泌作准备, 五龄第5天的基因表达主要是合成分泌丝素蛋白; H链、L链和P25蛋白实际可能并没有按6︰6︰1比率构成复合体, 或H链结构特殊, 不易通过EST技术检测到. 在2861个基因中将有相当部分基因参与丝素蛋白的合成与分泌, 说明家蚕后部丝腺细胞合成、分泌丝素蛋白这一生命过程比以前了解的要复杂得多.     

转基因家蚕丝腺组织和转化家蚕培养细胞表达hIGF-I

为实现在家蚕培养细胞和家蚕丝腺组织中表达人胰岛素样生长因子（hIGF-I）基因,以家蚕丝胶基因启动子（Pser-1）驱动hIGF-I基因,构建了带有家蚕杆状病毒ie-1启动子（Pie-1）控制的neo基因表达盒的转基因载体pigA3GFP—hIGF-ie—neo．在可表达转座酶的辅助质粒存在下,分别转染BmN培养细胞,以700～800μg/mL的G418筛选,获得了稳定转化细胞系．Westernblotting分析可检测到hIGF-I的特异性条带,ELISA检测结果显示,hIGF-I在5-100个细胞中的表达水平约7800Pg．通过反向PCR分析表明,在转化细胞中外源DNA可通过随机整合或按照piggyBac特定的靶位点序列TTAA插入细胞基因组．转基因载体pigA3GFP-hIGF—ie-neo通过精子介导法导入蚕卵,利用neo,加基因的双重筛选,经过PCR和点杂交鉴定,获得了2头转基因家蚕．ELISA检测结果显示,在G1代hIGF-I在每克中部丝腺组织中的表达水平为2440Pg左右．     

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.     

pH对红褐斑腿蝗中肠主要蛋白酶活性的影响

为评价pH对红褐斑腿蝗Catantops pinguis (Stål)中肠蛋白酶活性的影响, 本文用3种专性底物测定了不同pH环境下蝗虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性。结果表明: 雄性红褐斑腿蝗中肠肠液的pH值为6.92±0.043, 雌性为7.03±0.054, 两性间差异不显著（P>0.05）。并且发现3种蛋白酶的最适pH值各不相同, 其中雌雄虫的强碱性类胰蛋白酶（以BAPNA为底物）最适pH分别为8.5和10.5; 雌雄虫的弱碱性类胰蛋白酶（以TAME为底物）最适pH分别为9.0和9.5; 而雌雄虫的类胰凝乳蛋白酶（以BTEE为底物）最适pH雌性为8.5, 雄性为8.0。统计结果显示, pH对红褐斑腿蝗中肠蛋白酶活性影响显著（P<0.01）, 两性间蛋白酶活性差异显著（P<0.01）。在最适pH情况下, 雌性的类胰蛋白酶活性高于雄性, 而类胰凝乳蛋白酶活性则是雄性高于雌性。在中肠pH范围内雌性比雄性具有更高的消化蛋白酶活性, 显示雌性具有较强的食物处理能力以摄取更多的营养物质为繁殖活动（孕卵）作准备, 而该种蝗虫最适pH范围较宽, 可能与其取食植物范围较宽有关。     

Expression of the hIGF-I gene driven by the Fhx/P25 promoter in the silk glands of germline silkworm and transformed BmN cells

The expression of the human insulin-like growth factor (hIGF-I) gene driven by the Fhx/P25 promoter in the silk glands of transgenic silkworms (Bombyx mori) and in transformed silkworm cells, was achieved using BmN cells transfected with a piggyBac vector, pigA3GFP-Fhx/P25-hIGF-ie-neo containing a neomycin-resistance gene (neo), a green fluorescent protein gene (gfp), an hIGF-I gene, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. We selected stably transformed BmN cells expressing hIGF-I using the antibiotic G418. The expression level of hIGF-I was about 450 pg in 3 × 10(6) cells, determined by ELISA. The piggyBac vector was transferred into the silkworm eggs using sperm-mediated gene transfer. The expression level of hIGF-I per gram fresh posterior silk glands of G4 transgenic silkworms was approx. 150 ng.     

Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells

To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700—800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×10⁵ cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.     

A new method for the modification of fibroin heavy chain protein in the transgenic silkworm

We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3' region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A((21)) and N((22)). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm.     

家蚕生存素基因在BmN4细胞有丝分裂中的功能分析

【目的】阐明生存素基因在家蚕Bombyx mori BmN4细胞有丝分裂中的功能。【方法】qRT PCR分析家蚕生存素基因BmSurvivin在家蚕5龄第3天幼虫不同组织(丝腺、中肠、马氏管、精巢、卵巢、脂肪体、皮细胞层和表皮)中的表达量;构建pIZT/V5-His-BmSurvivin-GFP (BG)融合载体并转染BmN4细胞,以免疫荧光标记检测BmSurvivin和第10位丝氨酸(Ser10)磷酸化的组蛋白H3(H3Ser10ph)在BmN4细胞有丝分裂分裂不同时期的定位。【结果】BmSurvivin在家蚕5龄第3天幼虫马氏管中表达量最高,其次是在丝腺和中肠中。成功构建pIZT/V5-His-BmSurvivin-GFP载体。免疫荧光结果显示,在BmN4细胞间期核中可以看到明显的GFP信号,涵盖了细胞核和细胞质区域,指示BmSurvivin的定位;随着细胞进入分裂期,GFP信号指示BmSurvivin与染色质共定位,待细胞形成明显的双取向时在纺锤体区域也可以看到GFP信号;后期随着姐妹染色单体分开,GFP信号指示BmSurvivin定位在染色质及胞质分裂区;末期随着两个新的子细胞形成GFP信号指示BmSurvivin仅定位在胞质分裂区。H3Ser10ph定位在BmN4细胞前中期染色质凝缩处,至中期信号最强,与整条染色体重合,后期信号消失。【结论】BmSurvivin与有丝分裂周期中染色质和纺锤体的动态变化相关。     

Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein （egfp） gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part （up to 14 amino acid residues） of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide （mutant 13 aa）, the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue（s） in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue（s）. Furthermore, the deletion ofArg^2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.