酒类酒球菌苹果酸-乳酸酶基因的克隆

利用PCR方法从酒类酒球菌(Oenococcus oeni)基因组中扩增出651 bp的DNA片段,将之克隆到pUC19-T载体上并转化大肠杆菌(E.coli)JM109菌株.重组质粒的测序结果表明,克隆到了苹果酸-乳酸酶基因(mle),它含有527 bp的阅读框架,其核苷酸序列与文献报道相同.     

猪链球菌2型溶血素基因与38KDa蛋白基因克隆及联合表达

目的:研究有效的猪链球菌病疫苗。方法:以四川株猪链球菌2型基因组DNA为模板分别扩增溶血素基因和38KDa基因主要功能区基因;并经连接、克隆及酶切鉴定。分别构建原核表达载体pET32a-Sly、pET32a-38KDa,提取阳性克隆质粒分别进行双酶切并纯化,通过PCR串联两片断,将目的片断定向克隆到表达载体pET32a中,经测序正确后,重组质粒转化入大肠杆菌BL21 (DE3),用IPTG进行诱导表达。结果:重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子量约为60Ku的重组蛋白,表达产物经纯化后,免疫印迹法(Western-blotting)证实该重组蛋白可以与SS2阳性血清发生特异性反应。结论:本研究为重组蛋白的免疫保护应用奠定基础。     

家蝇抗菌肽Defensin基因同向串联表达载体的构建和鉴定

目的:构建家蝇抗菌肽Defensin基因多拷贝串联体,并克隆到甲醇酵母分泌表达载体pPIC9K上。方法:PCR法扩增家蝇抗菌肽Defensin基因成熟肽片断,目的片断的上游5′端带有EcoRⅠ和NheⅠ位点,下游5′端带有NotⅠ和XbaⅠ位点,目的片断首先克隆入pMD18-T载体,利用pMD18-T载体的NdeⅠ位点和目的片断上的一对同尾酶(NheⅠ和XbaⅠ),多次酶切连接,串联成多拷贝的Defensin成熟肽基因,再用EcoRⅠ和NotⅠ双酶切,最后克隆入甲醇酵母分泌表达载体pPIC9K。结果:PCR鉴定、酶切鉴定和DNA测序证明多拷贝基因重组质粒构建成功。结论:该方法能方便高效地获得所需的多拷贝基因,为进一步进行高效表达打下基础。     

棉铃虫核多角体病毒进化保守性基因iap2基因表达载体的构建及表达

分析棉铃虫核多角体病毒基因组 ,结合GenBank中已知的序列 ,发现iap2基因位于其基因组的BamHⅠ F片段上 ,回收此片段作为模板 ,设计引物 ,通过PCR扩增得到了抗细胞凋亡基因iap2的DNA片段。将扩增产物克隆到pGEM T载体上 ,再进一步将插入片段酶切并连接到表达载体pET 2 8a上 ,构建了重组质粒pET iap2。DNA序列分析结果表明 ,克隆得到的DNA序列与所发表序列完全相同。含重组质粒pET iap2的大肠杆菌BL2 1 (DE3)表达了抗细胞凋亡蛋白IAP2。     

乳酸克鲁维酵母表达型T载体的研发及应用

[目的]构建以乳酸克鲁维酵母表达载体p KLAC2为基础的表达型T载体。[方法]利用定点突变技术突变载体p KLAC2上的XcmⅠ位点,获得表达载体p KL-MUT;PCR扩增含黄色荧光蛋白基因的XcmⅠ酶切盒,通过EcoRⅠ和XhoⅠ位点连接至表达载体p KL-MUT,构建成重组载体p KL-YFP,限制性内切酶XcmⅠ酶切后即产生T载体p KL-T。连接融合基因14-3-3-Zs G转化至乳酸克鲁维酵母GG799。[结果]利用该T载体可成功克隆外源基因14-3-3-Zs G并在乳酸克鲁维酵母中表达14-3-3-Zs G蛋白,荧光和Western blotting验证正确。[结论]乳酸克鲁维酵母表达型T载体已成功构建,具有快速克隆高效分泌表达外源基因的特点,对于促进乳酸克鲁维酵母表达系统表达相关蛋白的产业化具有实际意义。     

米曲霉FleA基因的原核表达及纯化

目的研究米曲霉FleA基因克隆菌株的构建及米曲霉凝集素(Aspergillus oryzae lectin, AOL)蛋白表达。方法培养米曲霉RIB40,TRIzol法提取米曲霉RNA,用反转录试剂盒合成FleA全基因,设计引物扩增FleA基因,并将此片段与PJET克隆质粒连接构建克隆菌株,经双酶切筛选出阳性克隆菌株送去测序,将序列正确的克隆菌与表达载体pET-28a(+)分别经过双酶切后连接,转化到宿主菌E.coil BL21,IPTG诱导AOL目的蛋白表达,SDS-PAGE检测分析。结果 PCR成功扩增获得FleA基因,大小约950 bp,成功连接至PJET克隆载体后,经双酶切回收后又与pET-28a(+)成功连接,经过IPTG诱导后SDS-PAGE检测结果显示目的基因成功表达。结论该研究成功构建了米曲霉FleA基因,获得了表达蛋白,为今后结构和功能研究奠定了一定的基础。     

人干扰素α-2b基因植物表达载体的构建及转化

目的:通过构建人干扰素α-2b(hIFNα-2b)基因的植物表达载体,为后期将其导入胡萝卜愈伤组织中作准备。方法:采用PCR技术从人基因组DNA扩增hIFNα-2b编码基因及全长基因,将其克隆于pMD19-T载体中,双酶切hIFNα-2b基因及植物表达载体pBI121,回收目的片段,T4DNA连接酶连接得到植物表达载体pBI121-hIFN。采用三亲交配法将后者导入根瘤农杆菌。结果:经PCR检测,双酶切及DNA序列测定表明hIFNα-2b编码基因及全长基因已分别插入植物表达载体pBI121中,重组表达载体pBI121-IFN已成功转化根瘤农杆菌LBA 4404。结论:成功构建了植物表达载体pBI121-hIFN并转入根瘤农杆菌。     

果蝇dro基因的克隆与原核表达载体构建

目的:检测drosocin对农作物致病菌的抑菌作用,构建含drosocin基因dro的原核表达载体。方法:以黑腹果蝇(Drosophila melanogaster)DNA为模板,由特异引物通过PCR方法扩增dro基因的编码序列,将此片段连接在克隆载体pMD18-T上进行测序,再用酶切-连接的方法将目的片断亚克隆到携带有6×组氨酸二氢叶酸还原酶标签的原核表达载体pQE40上。结果:克隆得到大小为195bp的dro基因片段,并成功构建了原核表达载体pQE40/dro。结论:克隆到dro基因,构建了原核表达载体pQE40/dro,并获得了转化株M15[pREP4]/dro。     

携带人二氢叶酸还原酶基因慢病毒表达载体构建及鉴定

目的:构建携带人二氢叶酸还原酶(DHFR)基因的慢病毒表达载体pWPI。方法:采用PCR方法扩增二氢叶酸还原酶cDNA全长,与EZ-T克隆载体连接,HindIII及BamHI-HF限制性内切酶双酶切回收的PCR片段并补平其缺口。慢病毒系统载体使用pWPI系统,采用PmeI酶切载体后回收片段,将其磷酸化,T4酶连接载体与目的基因。表达载体鉴定均采用核苷酸序列测定,重组质粒采用脂质体转染293T包装细胞后获得包装的病毒颗粒。结果:成功扩增二氢叶酸还原酶全长并连接入pWPI载体构建成重组表达载体DHFR-pWPI,重组质粒测序结果显与DHFR基因的同源性达100%,按标准生产程序转染293T后有DHFR基因的表达。结论:成功采用慢病毒载体系统构建了二氢叶酸还原酶重组慢病毒转基因,为探讨DHFR在肿瘤多药耐药过程中的分子机理奠定基础。     

甲型副伤寒沙门菌PagC蛋白在乳酸乳球菌中的表达研究

目的构建能够表达甲型副伤寒沙门菌外膜蛋白(Pag C)的乳酸乳球菌。方法 PCR扩增pag C基因,亚克隆至p MD18-T载体并测序,从亚克隆载体中酶切回收pag C基因,插入p MG36e并命名为p MG36e-pag C,转化乳酸乳球菌MG1363。溶菌酶加超声的方法破碎重组乳酸乳球菌,SDS-PAGE以及Western blot检测Pag C蛋白的表达。结果 p MG36e-pag C鉴定正确,Western blot检测到p MG36e-pag C/MG1363表达的Pag C蛋白。结论成功构建能组成型表达Pag C蛋白的乳酸乳球菌。     

幽门螺杆菌热休克蛋白A在乳酸乳球菌中的表达及免疫学活性分析

目的构建含幽门螺杆菌(H.pylori)热休克蛋白A编码基因的重组载体,并电转入乳酸乳球菌MG1363,表达目的蛋白并分析其免疫原性,为H.pylori基因工程口服疫苗的研究和开发奠定基础。方法以H.py-loriNCTC 11637株基因组DNA为模板,PCR扩增hspA基因,并克隆至乳酸乳球菌表达载体pMG36e中。将重组质粒转化E.coliDH5α,经鉴定的阳性重组质粒命名为pMG36e/hspA。以电穿孔法将pMG36e/hspA转化乳酸乳球菌MG1363并用Western blot检测HspA蛋白的表达。结果克隆重组后得到pMG36e/hspA。将pMG36e/hspA电转化MG1363后,收集菌体蛋白进行Western blot分析,在HspA的相对分子质量(Mr≈13 kDa)处出现特异性条带。结论首次成功构建了表达H.pyloriHspA的重组乳酸乳球菌MG1363,为进一步口服疫苗的相关研究奠定了基础。     

人血清白蛋白在乳酸克鲁维酵母中的表达

以乳酸克鲁维酵母(Kluyveromyces lactis,K.lactis)GG799为宿主对人血清白蛋白(HSA)进行分泌表达。以pPIC9k-HSA为模板,采用带有XhoⅠ和NotⅠ酶切位点的引物PCR扩增获得HSA基因,经XhoⅠ和NotⅠ双酶切后插入pKLAC1,构建表达载体pKLAC1-HSA。经SalⅡ线性化后,电击转化K.lactis GG799,用含5 mmol/L乙酰胺的YCB平板筛选阳性转化子。提取基因组DNA,采用PCR方法对转化子鉴定后进行摇瓶发酵。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot分析发酵上清液中的表达产物,并初步分析酵母基础N源(YNB)对HSA在K.lactis GG799中表达的影响。结果表明,HSA成功在K.lactis GG799中分泌表达,表达量为81μg/mL,遗传稳定性好。     

Use of DNA quantification to measure growth and autolysis of Lactococcus and Propionibacterium spp. in mixed populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.     

Application of PCR, rep-PCR and RAPD techniques for typing ofLactococcus lactis strains

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.     

Rapid PCR-based method which can determine both phenotype and genotype of Lactococcus lactis subspecies

A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different L. lactis strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the L. lactis subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments.     

植物乳杆菌ZS2058的亚油酸异构酶基因在乳酸克鲁维酵母中的克隆表达

目的：将植物乳杆菌ZS2058（Lactobacillus plantarum ZS2058）的亚油酸异构酶基因在乳酸克鲁维酵母（Kluyveromyces lactis）中进行克隆表达。方法：根据NCBI中已报道亚油酸异构酶（linoleate isomerase,LAI）基因的序列特征,设计引物对筛得的植物乳杆菌ZS2058进行PCR扩增,得到亚油酸异构酶全基因序列,克隆至乳酸克鲁维酵母表达载体pKLAC1,电转化得重组菌pKLAC1-LAI /Kluyveromyces lactis GG799。结果：SDS-PAGE检测,重组菌进行分泌表达获得目的蛋白,大小约为67 kDa;气相色谱（Gas Chromatogram,GC）检测到共轭亚油酸（conjugated linoleic acids,CLA）典型峰。结论：植物乳杆菌ZS2058中的亚油酸异构酶基因在乳酸克鲁维酵母中得到分泌表达,重组酶转化效率约为26%。     

乳酸乳球菌AL2基因文库的构建及乳链菌肽生物合成基因簇的筛选

用本实验室获得的乳链菌肽高产菌株乳酸乳球菌AL2总DNA为供体,以λ EMBL3为载体,构建了该菌株的基因文库,共获得3900多个噬斑。通过Southern杂交、PCR扩增及DNA序列测定,证实从该文库中筛选到一个含有完整乳链菌肽生物合成基因簇的重组噬菌体λHJ\|3。     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

Rapid differentiation of the closely related Kluyveromyces lactis var. lactis and K. marxianus strains isolated from dairy products using selective media and PCR/RFLP of the rDNA non transcribed spacer 2

PCR/RFLP of the NTS2 (IGS2) of rDNA was applied to differentiate two closely related yeast species, Kluyveromyces lactis var. lactis (referred to as K. lactis) and K. marxianus. Using specific primers, the NTS2 region was amplified from DNA of both K. lactis and K. marxianus type and collection strains. AluI restriction of amplified fragments generated patterns characteristic for each species. The NTS2 region from K. lactis var. drosophilarum and related species K. aestuarii, K. africanus, K. dobzhanskii, and K. wickerhamii could also be amplified with the same primers, but AluI patterns generated were clearly different. PCR/RFLP of the NTS2 appears thus to be a convenient method for rapid identification of K. lactis and K. marxianus, frequently found in dairy products. This test was validated therefore on K. lactis and K. marxianus from natural habitats. We showed that all yeast strains collected from whey samples and scoring blue on X-gal glucose plates were either K. lactis or K. marxianus. For application purposes, we propose here an approach for quickly screening for K. lactis/marxianus and Saccharomyces cerevisiae in dairy products using X-gal coloured and lysine growth media.     

Mutations in Mgi Genes Convert Kluyveromyces Lactis into a Petite-Positive Yeast

Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.