利用绿色荧光蛋白基因gfp研究芽胞杆菌的启动子活性

利用绿色荧光蛋白基因gfpmut3,分别标记苏云金芽胞杆菌(Bacillus thuringiensis)的cry3A启动子Pcry3A、BtI_BtII启动子PBtI_BtII和来自蜡状芽胞杆菌特异启动子P44-12以研究其表达差异。其中,Pcry3A和PBtI_BtII分别与gfpmut3构成融合基因,以调控gfpmut3在苏云金芽胞杆菌中的表达。将重组质粒pGFP_304(含P44-12)、pGFPExpA(含Pcry3A_ gfpmut3融合基因)和pGFPExpB(含PBtI_BtII_ gfpmut3融合基因)分别导入大肠杆菌(Escherichia coli)和苏云金芽胞杆菌后发现,P44-12和PBtI_BtII在大肠杆菌与苏云金芽胞杆菌中均可表达gfpmut3,其中PBtI_BtII在大肠杆菌中具有极强的启动基因表达的能力。而Pcry3A不能启动gfpmut3在大肠杆菌中表达,在苏云金芽胞杆菌中启动的gfpmut3表达的荧光强度也较弱。进一步通过荧光显微镜和生物活性检测器对含重组质粒pGFP_304、pGFPExpA和pGFPExpB的转化子分别进行荧光检测及微量热检测。结果表明,3种启动子驱动下的gfpmut3基因均可在苏云金芽胞杆菌无晶体突变株BMB171中表达并检测得到不同的发光类型。微量热法检测发现P44_12和PBtI_BtII启动gfpmut3表达的代谢热低于Pcry3A驱动gfpmut3表达的代谢热。     

苏云金芽孢杆菌分子伴侣串联基因p19-p29的克隆和表达载体的构建

根据已知序列设计一对PCR引物(ORF5S,ORF3N),可从cry2Aa或cry2Ac操纵子中扩增出包含串联分子伴侣基因p19p29的DNA片段,预期大小分别为16kb和20kb。对150株苏云金芽孢杆菌菌株进行PCR检测,从26株中获得了大小为16kb的扩增片段,但未获得20kb的片段。这表明cry2Aa型操纵子p19p29基因存在较广泛,而cry2Ac型较罕见。将来自Y2菌株的16kb片段回收,通过一系列亚克隆,最终构建成一个含有p19p29串联基因的Bt表达载体,为进一步研究p19p29串联基因的功能奠定了基础。     

苏云金杆菌辅助蛋白P20对杀虫晶体蛋白Cry1Ab表达的影响

苏云金杆菌(Bacillus thuringiensis, 简称Bt)杀虫晶体蛋白Cry1Ab因其C半端缺少了一段含4个半胱氨酸的氨基酸序列而导致蛋白的不稳定,报道苏云金杆菌辅助蛋白P20帮助Cry1Ab蛋白的表达及晶体的形成。利用穿梭载体pHT3101构建3个表达质粒,即pT1B、pP1B和pDP1B,3个质粒都含有cry1Ab基因,不同在于pT1B没有p20基因,pP1B含有p20全基因,而pDP1B不仅含有p20全基因,且在p20基因前插入cry1A?启动子。分别将这3个表达质粒经电转化到苏云金杆菌晶体缺陷型菌株CryB中,获得转化菌株T1B、P1B和DP1B。Western blot表明cry1Ab基因在这3株菌中均表达了130 kD的蛋白,部分降解为大约60 kD的蛋白。蛋白定量分析显示,3株菌130 kD蛋白量的比为1∶1.4∶1.5,降解后的60 kD蛋白量的 比为1∶1.1∶1.6,Cry1Ab蛋白总量的比为1∶1∶2∶1.6。镜检发现,Cry1Ab在3株菌中都形成典型的菱形晶体,其晶体大小为T1B Helicoverpa armigera）均具有明显的杀虫活性,三者的LC₅₀差异不显著。研究表明,P20对cry1Ab基因的表达和晶体形成均有帮助,P20表达量的多少可能是导致Cry1Ab蛋白最终产量有所不同的因素。     

苏云金芽胞杆菌杀虫晶体蛋白基因cry1Ab16的克隆及表达

通过对已知cry1类基因以及已发表的cry1Ab的序列进行分析,分别设计了引物P1、P2、P3和P4,首次从无晶体的芽胞杆菌AC11中扩增到一个苏云金芽胞杆菌杀虫晶体蛋白（Insecticidal crystal protein, ICP）cry1Ab类基因。测序结果显示该基因与已知的cry1Ab1基因有8个核苷酸不同,编码的蛋白有7个氨基酸差异。此基因已登录GenBank,并命名为新亚型基因cry1Ab16 (Ac. NO. AF375608)。Southern杂交结果进一步证实该基因存在于菌体的质粒上。将cry1Ab16基因克隆到Escherichia coli表达载体pQE30上并转化E. coli M15。Western印迹分析表明,E. coli M15表达了130 kD的Cry1Ab16蛋白,但此蛋白不稳定,大部分降解成65 kD的蛋白。将表达Cry1Ab16 蛋白的大肠杆菌用涂布法对三龄小菜蛾（Plutella xylostella）毒力测定,其LC50为258.3mg/L;对其他夜蛾科害虫的生长发育也有明显的抑制作用。     

华葵根瘤菌nifA基因的克隆和功能分析

华葵根瘤菌(Mesorhizobium huakuii即R.astragali)159的nifA基因的序列分析表明,该基因全长1227bp,编码分子量为44734D的Nif A蛋白。与其它NifA蛋白的序列比较发现,华葵根瘤菌NifA蛋白也存在保守的中间结构域和C末端DNA结合结构域,但其氨基端缺失。Tn5定点突变得到的突变体是Nif-表型。构建了nifA基因组成型表达的质粒,此质粒在大肠杆菌中对华葵根瘤菌nifHlacZ有激活作用。     

大肠杆菌\%otsA\%基因的克隆和表达

用PCR方法扩增了15kb的otsA基因片段,将该片段连接到多拷贝克隆载体后转化otsBA缺失和otsA缺陷的大肠杆菌菌株,使转化株重新获得otsA基因功能。生长曲线表明转化株在高渗培养基中生长良好,薄层层析法(TLC)检测海藻糖实验说明转化株细胞经诱导后合成海藻糖,otsA基因的克隆和表达为赋予转基因植物抗高渗、耐干旱能力提供了实验依据和材料。     

苏云金芽胞杆菌YBT1520杀虫晶体蛋白基因的属性

通过Southern杂交发现高毒力苏云金芽胞杆菌(\%Bacillus thuringiensis)\% YBT1520菌株含有两个杀虫晶体蛋白基因片段,其5’末端所在HindⅢ片段分别为68kb和46kb,它们对应的基因分别命名为cry218和cry46。经PCR鉴定,该菌含有cry1Aa\,cry1Ab和cry1Ac基因,以及cry2基因,其中cry218属于cry1Ac。分析了cry218基因4190bp的核苷酸序列,在杀虫晶体蛋白基因分类系统中被命名为cry1Ac10。结合Southern杂交和PCR结果可判断3个cry1A基因的拷贝数不同,其中cry1Ac拷贝数最高,YBT1520菌株与其它库斯塔克亚种的杀虫晶体蛋白基因所在限制性内切酶位置明显不同。     

苏云金芽孢杆菌科默尔亚种15A3株的cry基因分析及杀虫特性

筛选的苏云金芽孢杆菌野生菌株15A3经鉴定属血清H21型科默尔亚种。用PCR及RFLP方法对其cry1类基因分析证明其含有cry1Aa,\%cry1\%Ac,\%cry1\%Ca,\%cry1\%D,\%cry1\%I及cry2六种cry基因,其cry1A基因N末端145kb片段与已发表的序列有差异。表达晶体蛋白质的分子量分别为130,79,70,65,51和45kD。对家蝇致畸实验证明其不含β外毒素。发酵液对棉铃虫,甜菜夜蛾,小菜蛾及美国白蛾均具较高的毒力。证明野生的苏云金芽孢杆菌资源中也有具国外工程菌所特有的高效杀虫晶体蛋白基因组合的优良菌株。     

Sinorhizobium morelensS-5中N_氨甲酰基水解酶基因的克隆、表达和纯化

N_氨甲酰基水解酶是一种非常具有工业应用价值的水解酶,可用于制备光学纯氨基酸。通过LA PCR 从 Sinorhizobium morelens S_5 菌中克隆到13kb的DNA片段,测序表明该片段上含有一个完整的N_氨甲酰基水解酶的基因（hyuC）序列。将hyuC基因克隆到表达载体pET30a上,重组质粒pET30a_HyuC在大肠杆菌中获得了高水平表达。重组的N_氨甲酰基水解酶经过热处理和三步柱色谱分离而纯化。纯化倍数为16.1倍,收率21.2%。该酶为同源四聚体,亚基分子量是38kDa。最适温度是60℃,最适pH为7.0。该酶有较高的热稳定性和氧化稳定性。Fe²⁺和Ca²⁺对酶的活性有一定的促进作用,而金属螯合剂和巯基试剂对酶活无明显影响。     

苏云金芽胞杆菌以色列亚种20kDa蛋白质对CytA蛋白溶细胞作用的影响

为了证明苏云金芽胞杆菌以色列亚种２０ｋＤｅ蛋白质对ＣｙｔＡ蛋白溶细胞作用的影响, 根据２０ｋＤｅ蛋白质和ｃｙｔＡ蛋白基因的核苷酸序列,用ＡＭＰＬＩＦＹ程序设计了一套带有酶切位 点的引物,经ＰＣＲ扩增分别获得了２０ｋＤｅ蛋白质和ｃｙｔＡ蛋白基因。将其基因与表达载体 ｐＵＨＥ２４连接并转化到大肠杆菌ＸＬＩ和ＤＨＳ 分别获得含２０ｋＤａ蛋白质基因的克隆子 ＬＺ２９;含ｃｙｔＡ基因的克隆子ＬＺｃｙｔＡ和含有二者基因的重组子ＬＺ２０Ａ．在ＩＰＴＧ诱导下,测定 了不同克隆株基因表达产物对大肠杆菌细胞生长的影响。结果表明：ＬＺ２０的细胞生长不受影 响;ＬＺｃｙｔＡ的细胞被杀死;ＬＺ２０Ａ的细胞生长也不受影响。这表明２０ｋＤａ蛋白质基因与ｃｙｔＡ 蛋白基因重组后,２０ｋＤａ蛋白质基因表达产物可保护ＣｙｔＡ蛋白对大肠杆菌的溶细胞作用,而 巳这种作用并不因不同大肠杆菌受体而改变。     

肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中克隆的研究

在大肠杆菌中克隆肺炎支原体P1蛋白羧基端基因片段,为P1蛋白基因片段的扩增、表达及探讨羧基端基因片段功能打基础.采用PCR扩增方法获取P1结构基因.扩增产物用SalI和EcoRI酶切消化,回收1kb大小的DNA片段并与pUC19DNA连接,转入大肠杆菌JM109菌株.用X-gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定.经PCR扩增MPDNA获得1条5.0kbDNA片段.重组质粒限制性内切酶指纹图谱显示出2条带,1条为pUC19载体DNA带,另1条是1kb的插入片段.实验获得肺炎支原体P1蛋白结构基因及含P1蛋白羧基端DNA片段的重组克隆株.     

酿酒酵母海藻糖合成酶基因的克隆和在大肠村菌中的表达

用PCR方法克隆了1．5kb的酿酒母Sacchromyces cerevisiae海藻糖合成酶基因TPSI,将该片段连接到pUC19载体,通过转化分别引入海藻糖合成酶基因缺失和缺陷的大肠杆菌Escherichia coli FF4169 和FF4050,对转化株的质粒DNA酶切分析表明均含有1．5kb PCR克隆片段,生长曲线实验证明,带有克隆片段的转化株在含0．5mol/L NaCl的高渗透压基础培养基中生长良好;用高效液相色谱（HPLC）结合蒸发散射（ELSD）技术测定细胞内海藻糖实验证明转化株能够合成海藻糖。     

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12.

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.     

乙型肝炎病毒C蛋白基因的克隆表达

根据已知的C蛋白基因序列设计一对引物,用PCR法从HBV adw亚型全基因组克隆中扩增出约500bp的NDA片段,将该基因克隆到表达质粒pQE30中,转化大肠杆菌,进一步对重组质粒中的插入片段进行DNA测序,证明是C基因,阳性克隆子经IPTG诱导后,获得了目的蛋白的表达,菌体经超声破碎后,目的蛋白主要存在于上清中,为下一步研究其抗原性和免疫原性奠定了基础。     

Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins.

Adenovirus mutants containing genetic alterations in the gene encoding the E1B 19,000-molecular-weight (19K) tumor antigen induce the degradation of host cell chromosomal DNA (deg phenotype) and enhanced cytopathic effect (cyt phenotype) after infection of HeLa and KB cells. The deg and cyt phenotypes are a consequence of viral early gene expression in the absence of the E1B 19K protein. The role of the E1A proteins in induction of the cyt and deg phenotypes was investigated by constructing E1A-E1B double mutant viruses. Viruses were constructed to express the individual E1A 13S, 12S, or 9S cDNA genes in the presence of a mutation in the gene encoding the E1B 19K tumor antigen. Expression of either the 13S or 12S E1A proteins in the absence of functional E1B 19K protein produced the deg and cyt phenotypes. In contrast, a virus which expressed exclusively the 9S E1A gene product in the absence of the E1B 19K gene product did not induce the deg and cyt phenotypes, even at high multiplicities of infection. Therefore, both the 13S and 12S E1A gene products could directly or indirectly cause the deg and cyt phenotypes during infection of HeLa cells with an E1B 19K gene mutant virus. Furthermore, the deg phenotype was found to be host cell type specific, occurring in HeLa and KB cells but not in growth-arrested human WI38 cells. These results indicate that expression of the E1A trans-activating and transforming proteins is necessary for the induction of the cyt and deg phenotypes and that host cell factors also play a role.