枯草芽孢杆菌中性蛋白酶基因的克隆与表达

本研究对枯草芽孢杆菌中性蛋白酶的基因进行克隆,并在大肠杆菌中诱导表达。从枯草芽孢杆菌基因组克隆到编码中性蛋白酶的全长基因,构建大肠杆菌原核表达载体pET30b,转化大肠杆菌BL21得到重组工程菌。通过菌落PCR和酶切筛选验证重组体。在25℃,110 r/min条件下,用IPTG诱导重组蛋白表达。表达蛋白用SDS-PAGE验证,并对酶活力进行测定。测序结果表明克隆序列与NCBI上的登录的序列同源性高达99%。SDS-PAGE结果表明诱导出的蛋白相对分子质量56.6 kD,酶活力达到39 U/mL。证明成功克隆得到枯草芽孢杆菌中性蛋白酶基因,并在大肠杆菌中得到高效表达。     

大肠杆菌葡萄糖异构酶基因在变铅青链霉菌中的克隆与表达

本文用穿梭质粒载体pSE-3,进行了大肠杆菌葡萄糖异构酶基因在变铅青链霉菌中的克隆与表达。把含有1.6kb葡萄糖异构酶基因的pX1200(4.3kb)质粒与pGEM-3(2.9kb)质粒分别用EcoRI酶解,T4DNA连接酶连接,转化E.Coli HB101(Xy1~-,Neo(?)),得到的重组质粒被命名为pX1203(7.2kb);将pX1203与穿梭质粒载体pSE-3分别用HindⅢ酶解,T_4DNA连接酶连接,转化E.Coli HB101,在含有新霉素的木糖培养基上筛选到转化重组体,其重组质粒被命名为pSE×100(10.6kb);把pSE×100转化变铅青链霉菌TK54的原生质体,在硫链丝菌肽(50μg/ml)和新霉素(50μg/ml)的平皿上得到了重组体。经质粒提取,酶切分析,再转化和葡萄糖异构酶活性测定,结果表明,大肠杆菌的葡萄糖异构酶基因确实已在链霉菌中克隆和表达。     

普通变形杆菌位置非特异性脂肪酶基因的克隆及在大肠杆菌中的表达

【目的】本文拟克隆普通变形杆菌(Proteus vulgaris)脂肪酶基因,并实现其在大肠杆菌中的高效表达,并检验外源表达脂肪酶的催化性质。【方法】通过PCR方法,从P.vulgaris基因组中扩增其脂肪酶基因(PVL),并将其开放读码框区域连接到表达载体pET-DsbA及pMBP-P上,在大肠杆菌中用IPTG诱导表达。我们对培养基组分及培养条件进行优化,以获得最高的酶产量。用Ni柱亲和层析法对所得重组脂肪酶进行纯化,并考察其酶学性质。【结果】PVL基因编码区含864个碱基,编码含287个氨基酸的酶蛋白。该序列在GenBank的登录号为FJ643627。PVL基因在大肠杆菌BL21内诱导表达的最佳条件为:在pH8.5的LB培养基中添加15g/L葡萄糖及200mg/L氨苄青霉素,在培养至OD600为1.2时加入100mg/LIPTG,15℃诱导15h,最高酶活达到192.2U/mL。通过亲和层析纯化了重组脂肪酶,得到一个约31kDa的蛋白条带。外源表达的脂肪酶的催化特性与野生菌脂肪酶相似,具有催化的位置非特异性,对长链脂肪酸酯亲和性最高。【结论】PVL基因在大肠杆菌中的高效表达为P.vulgaris脂肪酶的进一步研究与应用奠定基础。     

不同条件下两株溶磷菌溶磷量及葡萄糖脱氢酶基因表达与酶活分析北大核心CSCD

【目的】获得大豆根际土壤中溶磷能力较强的菌株,明确在菌株溶磷过程中葡萄糖脱氢酶(GDH)的作用特点及其基因的表达水平。【方法】利用溶磷圈方法分离与纯化溶磷菌株,采用Vitek 2系统和16S r RNA序列分析菌株的分类地位;测定2菌株的溶磷量、GDH活性,并根据GDH基因的保守区序列设计引物,克隆GDH基因,利用实时荧光定量PCR测定不同条件下基因的相对表达量。【结果】筛选出2株具有较强溶磷能力的溶磷菌,分别鉴定为Pseudomonas sp.和Enterobacter sp.,2菌株最高溶磷量分别为558μg/m L和478μg/m L;成功地克隆了2株溶磷菌的GDH基因,片段大小分别为2007 bp和2066 bp;2菌株在不同磷源、不同p H值培养基中GDH活性及基因表达量不同,菌株wj1在高磷条件下基因表达量最高,磷胁迫条件下基因表达量较低,而wj3在不同磷源条件下GDH基因表达量都较低。且GDH基因表达量及酶活的变化与wj3菌株溶磷量没有直接的关系。【结论】从大豆根际土壤中分离获得溶磷能力较强的菌株Pseudomonas sp.wj1和Enterobacter sp.Wj3,GDH活性及基因表达在2株菌溶磷过程中具有不同的作用特点,2菌株溶磷机制不完全相同。     

精氨酸脱亚胺酶在大肠杆菌中的分泌型表达

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arc A克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢB中,经鉴定得到重组质粒pBAD-ADI。将重组质粒转化大肠杆菌TOP10F'后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002%(w/v),25℃下诱导5 h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同)。采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL。SDS-PAGE分析显示,重组蛋白大小约为46 kD。     

类芦根际溶磷真菌的筛选、鉴定及其溶磷能力分析

为揭示类芦(Neyraudia reynaudiana)等水土保持植物的耐低磷机制,开发溶磷菌种质资源,提高赤红壤磷素利用率,从类芦根际土壤中筛选到一株溶磷能力较强的真菌FP1,经形态学和ITS序列分析,鉴定为黑曲霉(Aspergillus niger)。3种难溶性磷酸盐液体培养基接种菌株FP1后,其pH值和溶磷量的动态变化显示,培养液的pH值均呈显著下降趋势。溶磷量与培养时间有关,除磷酸三钙外,菌株FP1对其他难溶性磷酸盐的溶磷趋势均为先上升再下降并趋于稳定。菌株FP1对不同磷源的最大溶磷率顺序为:磷酸铝(92.02%)磷酸三钙(41.62%)3种磷酸盐的混合物(35.86%)磷酸铁(19.20%)。FP1对磷酸铝和磷酸铁都具有较强的溶磷能力,表明抗逆性强的水土保持植物类芦根际土壤蕴藏着高效的溶磷微生物资源。     

Secretory expression of arginine deiminase in E.coli

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arcA克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢ B中,经鉴定得到重组质粒pBAD-ADI.将重组质粒转化大肠杆菌TOP10F’后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002％ (w/v),25℃下诱导5h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同).采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL.SDS-PAGE分析显示,重组蛋白大小约为46 kD.     

嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a( )上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。     

斑马鱼尿酸氧化酶基因的克隆与原核表达

目的探讨斑马鱼尿酸氧化酶基因的克隆与原核表达的方法,研究重组斑马鱼尿酸氧化酶(ZUO)的理化性质与生物学功能。方法用PCR的方法克隆斑马鱼尿酸氧化酶基因,用pET28a质粒进行原核表达。结果成功克隆了斑马鱼尿酸氧化酶基因并完成了测序鉴定,该基因在大肠杆菌表达系统中得到有效表达且具有体外氧化尿酸的生物学功能。目的蛋白为胞内可溶性蛋白,占可溶蛋白总量的50%左右。最佳诱导条件为27℃16 h,粗酶最佳溶解pH为9.16,在此缓冲液溶解条件下,粗酶比活性为1.94 U/mg。获得的重组ZUO产量达64.8 U/g湿菌,高于直接从动物肝脏组织萃取的尿酸氧化酶。结论斑马鱼尿酸酶基因能在大肠杆菌表达系统中表达,其溶解pH比基因工程表达的重组猪尿酸氧化酶略低,对后续开发痛风治疗新药有利。     

双功能枯草杆菌诱导型高效表达分泌载体的构建与鉴定

利用大肠杆菌质粒pSP72和枯草杆菌质粒pUB18共整合得到双功能克隆载体pSB。在pSB多克隆位点依次引入枯草杆菌果聚糖蔗糖酶基因启动子-信号肽序列sacBp.s.、地衣芽孢杆菌淀粉酶基因终止子序列α-amyT和短小芽孢杆菌增强子基因degQ,最终构建了双功能枯草杆菌诱导型高效表达分泌载体pSBPTQ。将VasostatinⅠ基因作为靶基因检测sacBp.s.、α-amyT和degQ在pSBPTQ进行外源基因表达时的功能,结果表明,在蔗糖诱导下,sacB启动子有效启动了Vasostatin I基因的表达和分泌,α-amy T提高了VasostatinⅠ基因的转录效率,而degQ明显增强了VasostatinⅠ基因的表达水平。VasostatinⅠ基因在蔗糖诱导下成功表达并分泌到枯草杆菌细胞外,蛋白质分泌效率达到90%左右。质粒稳定性试验结果表明,经过40个世代之后,质粒pSBPTQ在枯草杆菌DB1342中仍旧保持在83%以上。     

运用生色基因标记黄瓜根围促生菌(PGPR)筛选菌株

采用三亲交配方法 ,通过Tn7转座系统将lacZY标记基因导入黄瓜根围促生菌 (PG PR)筛选菌株PseudomonasfluorescensCN1 1 6和PseudomonascorrugataCN31的利福平抗性突变株中 ;标记假单胞菌菌株则被赋予了利用乳糖作为唯一碳源的能力 ,在只有乳糖的M9培养基上生长能分解X Gal,菌落显出特有的蓝色 ;经Southern杂交分析 ,证明标记基因lacZY存在于转化菌株的染色体上 ;经验证标记菌株标记性状稳定 ,与对应的野生菌株比较其它性状如培养性状、形态特征、生防效果等基本不变 ;PGPR菌株利福平抗性和生色基因标记的结合 (双标记 )能最大限度地将土壤中引入的PGPR菌株与土著细菌分开 ,检测下限可达 1 0CFU mL ,为PGPR在根围的分子生态学研究提供了一个较好的工具。     

Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.     

Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis.

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.     

甘草提取物通过调控miR-212-5p/BCL2L2 基因抑制甲状腺肿瘤细胞的增殖、迁移和侵袭

目的探讨甘草提取物GL-1对甲状腺肿瘤细胞增殖、迁移和侵袭的影响及其分子机制。方法以10、20、30 μg/mL GL-1处理甲状腺肿瘤细胞CAL-62,或在CAL-62细胞中转染miR-212-5p mimics、anti-miR-212-5p、si-BCL2L2、pcDNA-BCL2L2。其中转染pcDNA-BCL2L2细胞并以30 μg/mL GL-1处理。噻唑蓝比色法 (MTT)检测CAL-62细胞增殖,Transwell小室法检测CAL-62细胞迁移和侵袭,实时定量PCR (qPCR)检测CAL-62细胞中miR-212-5p表达,Western blot检测相关蛋白Bcl-2样蛋白2 (BCL2L2)、细胞周期蛋白D1 (Cyclin D1)和基质金属蛋白酶-2 (MMP-2)表达。生物学信息预测miR-212-5p的下游靶基因,双荧光素酶基因报告实验进一步验证。数据采用单因素方差分析、Tukey’s事后检验和t检验。结果与对照组相比,10、20、30 μg/mL浓度GL-1降低CAL-62细胞24、48、72 h的细胞活性 (P < 0.05),并呈剂量、时间依赖性。与对照组相比,10、20、30 μg/mL浓度GL-1干预后,CAL-62细胞侵袭数[(143.56±14.22)个、(100.32±10.23)个、(68.23±6.49)个比(189.65±15.23)个]、迁移数[(198.56±14.35)个、(141.35±12.58)个、(89.56±8.95)个比 (295.36±17.56)个]和BCL2L2蛋白表达量 (0.76±0.08、0.51±0.06、0.24±0.02比1.00±0.12)均降低 (P 均< 0.05),而miR-212-5p水平 (1.61±0.11、1.99±0.13、2.28±0.15比1.00±0.07)升高(P < 0.05),并呈剂量依赖性。过表达miR-212-5p和沉默BCL2L2表达在24、48、72 h时CAL-62细胞活性、细胞迁移数、侵袭数和Cyclin D1、MMP-2蛋白表达量降低 (P < 0.05)。生物学信息预测和双荧光素酶基因报告实验证实BCL2L2是miR-212-5p的靶基因。过表达miR-212-5p抑制BCL2L2蛋白水平,沉默miR-212-5p促进BCL2L2蛋白表达 (P < 0.05)。过表达BCL2L2可逆转GL-1对CAL-62细胞增殖、迁移、侵袭及Cyclin D1、MMP-2蛋白表达的抑制作用。结论 GL-1通过miR-212-5p/BCL2L2抑制甲状腺肿瘤细胞的增殖、迁移和侵袭。     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌(Deinococcus radiodurans)recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E.coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D.radiodurans的recA基因在E.coli细胞中的表达能够完全补偿recA缺损E.coli辐射抗性能力。     

蛇毒锯鳞蝰素融合蛋白的发酵与纯化

研究大肠杆菌表达重组蛇毒锯鳞蝰素(Echistatin,Ecs)融合蛋白的发酵和纯化工艺。将Ecs基因插入表达载体pTXB1,转化E.coliBL21(DE3)构建工程菌。对工程菌进行补料分批培养并诱导表达,研究培养基、培养和诱导时间对工程菌生长和目的蛋白表达的影响,几丁质亲和层析纯化Ecs融合蛋白,经DTT裂解后,检测Ecs活性。发酵后菌体湿重可达75g/L,融合蛋白表达量约占总蛋白的35%,重组质粒在BL21宿主菌中传代稳定。亲和层析纯化后,得到Ecs单体,得率为28mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。优化了Ecs融合基因工程菌的发酵和纯化条件,为规模化生产奠定基础。     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli.

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.