乳酸乳球菌表面展示表达系统的构建及其鉴定

目的:旨在构建一个将抗原靶向于乳酸乳球菌细胞表面的表达系统。方法:运用PCR技术从金黄色葡萄球菌基因组中克隆出蛋白A(SPA)C-末端544个碱基对的锚定域序列(Spax)。通过酶切、连接将Spax构建入分泌型质粒pAMJ399形成携有整合外源基因位点BglⅡ的pAMJ400质粒。将报告蛋白—绿色荧光蛋白的基因(Gfp)插入载体pAMJ400的整合位点产生模式质粒pAMJ401并电转化其于乳酸乳球菌MG1363。绘制转化子MG1363(pAMJ401)生长曲线确认诱导期。调节pH值(6.0~6.5)诱导转化子并在荧光显微镜下观察杂合蛋白(GFP:SPAX)的表达情况。结果:在395nm的蓝色激发光下,诱导后的细菌发出较明亮的绿色荧光,而未诱导的细菌几乎不产生荧光。结论:成功地构建了乳酸乳球菌表面展示表达系统,此系统可以作为口服活菌疫菌研究的可行性操作平台。     

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。     

乳酸乳球菌组成型表面展示载体的构建及鉴定

目的探索融合有Usp45信号肽和3Lys M锚定序列的EGFP蛋白能否通过p32启动子组成型展示于乳酸乳球菌MG1363表面。方法融合PCR法分别将p32启动子与Usp45信号肽、3Lys M与egfp基因融合亚克隆至p MD-18T载体,鉴定正确后命名为18T-p Usp45、18T-3Lys M-egfp,分别将上述片段切下依次插入p MG36e构建p MG36e-p Usp45-3Lys M-egfp。将其转化入乳酸乳球菌MG1363,荧光显微镜观察及SDSPAGE、Western-blot检测。结果成功构建重组菌p MG36e-p Usp45-3Lys M-egfp/MG1363,荧光显微镜、SDSPAGE和Western-blot检测表明重组蛋白能在MG1363中组成型表达,且分泌至胞外并锚定在细胞壁上。结论成功地构建了组成型表面展示载体p MG36e-p Usp45-3Lys M-egfp,重组蛋白能分泌至胞外且展示在MG1363细胞壁上。     

食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达

为构建乳酸乳球菌食品级分泌表达载体,通过PCR扩增质粒pMG36e的p32启动子片段及乳酸乳球菌MG1363未知分泌蛋白(Usp45)基因的核糖体结合位点、分泌信号肽和成熟肽前11个氨基酸的编码序列(SPusp45),克隆到食品级载体pSH91中,构建食品级分泌性表达载体pSQ;克隆报告基因金黄色葡萄球菌核酸酶(NucA)成熟肽的编码序列nucA到pSQ中分泌信号后,转化乳酸乳球菌MBP71,构建了乳酸乳球菌食品级分泌性表达系统L lactis/pSQ-nucA;通过TB-D法和酶谱法检测L lactis/pSQ-nucA的表达形式、表达量并与以前构建的L lactis/pSQZ-nucA系统表达能力进行比较,结果发现L lactis/pSQ-nucA能够分泌性表达NucA,分泌性表达的NucA量大约是胞内NucA的10倍;L lactis/pSQ-nucA的表达量高于lactis/pSQZ-nucA.为进一步目的蛋白的的分泌性表达及食品级疫苗的研制奠定了基础.     

基于冰核蛋白的乳酸菌表面展示系统的构建

[目的]验证来源于丁香假单胞菌的冰核蛋白在乳酸乳球菌表面展示外源蛋白的可能性.[方法]以绿色荧光蛋白(Green Fluorescence Protein,GFP)基因gfp为报告基因,以冰核蛋白基因的N末端和NC端作为展示单元,构建乳酸菌表面展示载体pHZ101和pHZ102,并转化大肠杆菌(Escherichia coli JM109和乳酸乳球菌(Lactococcus lactis)MG1363.[结果]荧光显微镜观察显示重组大肠杆菌和乳酸乳球菌均能检测到绿色荧光.Western blot结果表明GFP蛋白在重组大肠杆菌和乳酸乳球菌中均得到表达,并且INPN-GFP蛋白多数滞留于乳酸乳球菌细胞质内,而INPNC-GFP蛋白则大部分定位于乳酸乳球菌的细胞膜上.[结论]以上结果表明丁香假单胞菌的冰核蛋白能引导外源蛋白定位于乳酸乳球菌的细胞膜上,为乳酸菌表面展示系统的构建提供了新的方向.     

F_(212-489)-3LysM融合蛋白与乳酸乳球菌的体外混合展示

探索C端融合有锚定序列3Lys M的呼吸道合胞病毒F截短蛋白(F212-489),能否通过体外混合展示于乳酸乳球菌MG1363表面。采用PCR方法扩增f212-489及3lys M基因,分别亚克隆至p MD18-T载体,在亚克隆载体中酶切回收上述两片段,插入p ET28a构成p ET28a-f212-489-3lys M,酶切鉴定并测序正确后转化BL21(DE3),经IPTG诱导获得的包涵体蛋白进行溶解、复性,将复性后的F212-489-3Lys M与乳酸乳球菌MG1363体外混合,通过全菌ELISA(enzyme linked immuno sorbent assay)检测融合蛋白吸附菌体的情况。成功构建重组菌p ET28-f212-489-3lys M/BL21(DE3),该菌诱导后F212-489-3Lys M主要以包涵体形式表达,F212-489-3Lys M与MG1363混合后,经全菌ELISA检测,F212-489-3Lys M组OD450远高于对照组,且差异非常显著(P0.01)。证明经大肠杆菌表达的重组蛋白F212-489-3Lys M经体外混合可展示于乳酸乳球菌MG1363表面。     

人谷胱甘肽硫转移酶A1在乳酸乳球菌中的表达及活性研究

用RTPCR技术从人肝总RNA中分离扩增了人谷胱甘肽硫转移酶A1基因的cDNA序列,克隆至大肠杆菌表达质粒pET23b,采用蛋白表达筛查法及DNA测序证明该cDNA序列完全正确。重组质粒pET23bhgst转化大肠杆菌BL21(DE3),经IPTG诱导获得高效表达的可溶性hGSTA1产物,其表达量约为大肠杆菌可溶性总蛋白的40%。将hGSTA1cDNA亚克隆至乳酸乳球菌表达载体pMG36e,电穿孔法转化乳酸乳球菌MG1363获得hGSTA1乳酸乳球菌表达株。SDSPAGE及Western杂交分析表明该菌株表达预期大小的hGSTA1融合蛋白,经谷胱甘肽琼脂糖亲和层析纯化获得的hGSTA1蛋白具有较高的谷胱甘肽硫转移酶活性。具hGSTA1酶活性的乳酸乳球菌工程菌可望应用于研制防癌保健乳制品。     

带锚定基序3LysM的EGFP融合蛋白与乳酸乳球菌的体外混合展示

目的探索融合有锚定序列的EGFP蛋白,能否通过体外混合展示于乳酸乳球菌MG1363表面。方法采用融合PCR方法扩增带有锚定序列的egfp(3LysM-egfp),亚克隆至pMD18T载体,测序正确后,插入表达载体pET28a转化BL21(DE3)进行诱导表达、纯化,将纯化的3LysM-EGFP与乳酸乳球菌进行体外混合作用,荧光显微镜及Western-blot检测展示效果。结果成功构建表达重组菌pET28a-3LysM-egfp/BL21并诱导出可溶性3LysM-EGFP,纯化后蛋白纯度达到81.5%,荧光显微镜及Western-blot均检测到展示的目的蛋白。结论纯化后的3LysM-EGFP能在体外条件下展示在乳酸乳球菌表面。     

猪白细胞介素18成熟蛋白基因的克隆与表达

目的克隆猪白细胞介素18(pIL-18)成熟蛋白基因,并在植物乳杆菌(Lb.plantarum)NC8中进行表达。方法通过RT-PCR方法从猪脾脏细胞中扩增出pIL-18成熟蛋白基因,克隆到T载体pMD18-T后测序;将阳性基因片段克隆至大肠埃希菌-乳酸菌穿梭表达载体pSIP-409构建重组表达载体pSIP-409-IL-18,进行酶切和PCR鉴定;应用电穿孔技术将其转化至Lb.plantarum NC8中,经SppIP诱导表达后,进行SDS-PAGE及Western-blot分析。结果经测序,pIL-18成熟蛋白基因核苷酸长度为579 bp,编码193个核苷酸;酶切和PCR鉴定证明成功构建了重组表达载体pSIP-409-IL-18;SDS-PAGE及Western-blot分析表明重组菌表达了18 kD的融合蛋白,该重组蛋白可以与鼠抗猪IL-18多克隆抗体反应。结论成功克隆了pIL-18成熟蛋白基因,并获得有生物活性的pIL-18重组乳酸菌,为研制开发IL-18重组乳酸菌制剂奠定基础。     

Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.     

The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis

Regulation of pyruvate-formate lyase (PFL) activity in vivo plays a central role in the shift from homolactic to mixed-acid product formation observed during the growth of Lactococcus lactis on glucose and galactose, respectively. Characterisation of L. lactis MG1363 in anaerobic batch cultures showed that the specific in vivo activity (flux) of PFL was 4-fold higher in L. lactis cells grown with galactose, compared with cells grown with glucose. The change in the PFL flux correlated with the observed variation in the PFL enzyme level, i.e. the PFL enzyme level was 3.4-fold higher in L. lactis cells grown on galactose than in those grown on glucose. To investigate whether a variation in the level of PFL was responsible for the shift in pyruvate metabolism, L. lactis strains with altered expression of pfl were constructed. The pfl gene was expressed under the control of different constitutive promoters in L. lactis MG1363 and in the PFL-deficient strain CRM40. Strains with five different PFL levels were obtained. Variation in the PFL level markedly affected the resulting end-product formation in these strains. During growth on galactose, the flux towards mixed-acid products was to a great extent controlled by the PFL level. This demonstrates that a regulated PFL level plays a predominant role in the regulation of the metabolic shift from homolactic to mixed-acid product formation in L. lactis.     

Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine beta-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.     

Expression of green fluorescent protein in Lactococcus lactis

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.     

Requirement of autolytic activity for bacteriocin-induced lysis

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co(2+) and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmADelta1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmADelta1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.     

南极假丝酵母脂肪酶B(CALB)基因在乳酸乳球菌MG1363中的整合及表达

根据南极假丝酵母脂肪酶B (CALB)的基因序列将CALB基因进行TA克隆、酶切鉴定及测序后,亚克隆至大肠杆菌-乳酸乳球菌穿梭表达栽体pMG36e-Nisl中,构建重组表达栽体pMG36e-Nisl-CALB.设计特异性引物P3和P4,对重组质粒pMG36e-NisI-CALB进行红霉素抗性基因的敲除,以构建食品级表达载体pMG36N-CALB,后再将两种重组质粒分别电转化入乳酸乳球菌MG1363,以Nisin为选择压力,考察CALB在MG1363中的表达情况.结果显示,成功构建了表达载体pMG36e-NisI-CALB及pMG36N-CALB,两株重组菌在含有20 IU Nisir/mL的培养基中均生长情况良好,遗传性能稳定,且经水解圈鉴定,CALB能够进行活性表达.进一步研究发现,CALB基因整合到乳酸乳球菌MG1363染色体中.     

Diversity of oligopeptide transport specificity in Lactococcus lactis species. A tool to unravel the role of OppA in uptake specificity

The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in the L. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in L. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situ binding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis.