德氏乳杆菌保加利亚亚种β-半乳糖苷酶基因在大肠杆菌中的非融合表达

目的在大肠杆菌中非融合表达德氏乳杆菌保加利亚亚种β-半乳糖苷酶,为构建能高效表达β-半乳糖苷酶的食品级益生菌株奠定基础。方法选择德氏乳杆菌保加利亚亚种(1.1480和wch9901)β-半乳糖苷酶基因(lacZ)起始密码ATG上游-18bp到ATG下游1bp的一段包含SD位点和ATGA位点的序列为上游引物,PCR扩增lacZ,插入表达质粒pMG36e构建重组表达质粒。转化大肠杆菌,筛选阳性克隆,提取重组质粒进行酶切鉴定和测序,并测定转化菌β-半乳糖苷酶活性。结果重组质粒酶切鉴定合格;其中插入的外源片段序列与德氏乳杆菌保加利亚亚种lacZ标准序列符合率达99%以上;携带重组质粒pMG36e-lacZ1.1480的大肠杆菌DH5α酶活性为3.074U/mL,酶比活性为6.939U/mgpro;携带重组质粒pMG36e-lacZwch9901的大肠杆菌DH5α酶活性为4.755U/mL,酶比活性为8.537U/mgpro。结论成功在大肠杆菌中非融合表达了德氏乳杆菌保加利亚亚种β-半乳糖苷酶;本研究选择的SD位点和ATGA位点能在大肠杆菌中有效地引导蛋白的非融合表达。     

乳酸菌超氧化物歧化酶基因的克隆与特性分析

目的：克隆乳酸菌超氧化物歧化酶（SOD）基因并进行特性分析。方法：根据已报道的乳酸菌SOD基因序列,采用PCR技术获得SOD碱基序列,将所得的PCR产物插入克隆载体pMD-18T中,重组质粒经酶切,PCR鉴定及测序,获得的序列进行生物信息学分析。结果：成功克隆了SOD基因,序列分析表明,该SOD基因由621bp组成,编码206个氨基酸残基,蛋白分子量为23KD,等电点为4．96。结论：该蛋白氨基酸序列主要以α-螺旋为主。     

结核分枝杆菌的aceA基因的克隆、序列测定及生物信息学分析

目的克隆编码结核分枝杆菌异柠檬酸裂合酶的基因aceA(1915-1916),对其全序列进行测定,并以生物信息学方法和软件分析其生物学特性．为进一步研究该酶的功能提供信息资料。方法用PCR系统扩增出异柠檬酸裂合酶基因片段,将其插入载体pET28a,对其全序列进行测定;利用序列对比分析、蛋白氨基酸序列分析及结构预测软件进行生物信息学分析和模拟。结果克隆的基因序列与报道的序列完全一致,aceA与icl及其他编码异柠檬酸裂合酶基因同源性很高,具有保守氨基酸共有序列;初步模拟出了该蛋白的三级结构。结论本研究通过对aceA基因序列和氨基酸进行生物信息学分析,获取了该酶的信息特征．并与现有的报道结果进行对比分析。为分子生物学诊断、治疗和药靶筛选提供理论依据。     

弓形虫三磷酸核苷水解酶基因的克隆与序列分析

目的：克隆并分析我国刚地弓形虫三磷酸核苷水解酶（NTPase）的编码基因。方法：采用聚合酶链反应（PCR）扩增弓形虫RH株的NTPase基因,克隆入pGEM-TEasy载体,转化后挑取阳性克隆进行酶切与测序鉴定,并对获得的NTPase基因及推导的氨基酸序列进行生物信息学分析。结果：PCR扩增得到特异的弓形虫NTPase基因序列,测序结果表明,获得的弓形虫NTPase-Ⅱ基因全长1812bp,编码的603个氨基酸与Genebank报道的氨基酸序列同源性为100%。经DNAStar预测NTPase-Ⅱ存在6个潜在抗原表位。结论：成功克隆出序列正确的弓形虫NTPase-Ⅱ基因,为其相关研究奠定了基础。     

黄粉甲抗冻蛋白基因克隆及生物信息学分析

目的：获得黄粉甲抗冻蛋白基因afpTx及相关生物信息学资料.方法：从黄粉甲幼虫中提取总RNA,通过RT-PCR合成黄粉甲抗冻蛋白基因afpTx的cDNA片段,克隆入载体pMD19-T,进行测序分析.酶切后将其亚克隆入表达载体pET32a（＋）,构建表达质粒pET32a-afpTx,并转化到大肠杆菌DL21后提取质粒,双酶切鉴定.采用MEGA4.0,BioEdit5.0.6软件对本研究克隆的抗冻蛋白基因afpTx进行氨基酸序列同源性变异及进化分析.结果：测序结果afpTx的cDNA长度为336bp;编码112个氨基酸;酶切、电泳结果表明克隆和亚克隆获得成功.抗冻蛋白氨基酸序列相似性分析表明afpTx与GenBank上提交的23条黄粉甲抗冻蛋白的氨基酸序列平均一致性为88%;与11条赤翅甲抗冻蛋白氨基酸序列的平均一致性为67%,2种甲虫的平均一致性为63%.进化树分析结果显示黄粉甲与赤翅甲抗冻蛋白序列是同源序列.赤翅甲的序列趋异度显著大于黄粉甲抗冻蛋白基因序列.结论：成功克隆了本地黄粉甲的afpTx基因,该序列是GenBank上提交的黄粉甲与赤翅甲抗冻蛋白的同源序列.     

银杏苯丙氨酸解氨酶(PAL)的基因克隆

目的通过克隆银杏苯丙氨酸解氨酶(PAL)基因并深入研究其结构,利用生物技术改良叶用银杏品质.方法利用PAL氨基酸序列中的保守区设计的简并引物,应用PCR等分子克隆技术,首次成功地从银杏基因组中克隆出了PAL基因片断.结果及结论 PAL基因长度为1140bp,所得序列已登录在NCBI的GenBank中,核酸序列和推测的氨基酸序列的Accession分别为AY231176和AAO73468.     

结核分枝杆菌phoS2基因的克隆及序列分析

目的 扩增结核分枝杆菌phoS2基因,并将其克隆至质粒pGEMT中进行核苷酸序列特性分析.方法 以结核菌标准菌株H3TRv为模板,通过PCR技术扩增出结核分枝杆菌phoS2抗原基因,将其重组到pGEM-T载体后进行酶切鉴定及序列测定和生物信息学分析.结果 成功扩增出结核分枝杆菌phoS2抗原基因,测序表明该片段开放阅读框为927bp,与已发表基因核苷酸序列相比,同源性为81%,推导编码氨基酸序列同源性为87%.结论 成功克隆phoS2基因,为结核病原核表达及相关研究奠定了基础.     

结核分枝杆菌rps12基因的克隆及核酸序列的分析

目的扩增结核分枝杆菌rps12基因,并将其克隆至质粒pGEMT中进行核苷酸序列特性分析。方法以结核菌标准菌株H37Rv为模板,通过PCR技术扩增出结核分枝杆菌rps12抗原基因,将其重组到pGEM-T载体后进行酶切鉴定及序列测定和生物信息学分析。结果成功扩增出结核分枝杆菌rps12抗原基因,测序表明该片段开放阅读框由375bp组成,与已发表基因核苷酸序列相比,同源性为100%,推导编码氨基酸序列同源性为99%。结论成功克隆rps12基因,经DNA测序证实,该片段阅读框完整,该基因的获得为其原核表达及相关研究奠定了基础。     

炭疽杆菌保护性抗原基因芯片探针的制备

目的快速制备炭疽杆菌保护性抗原基因芯片探针。方法根据PubMed上公布的炭疽杆菌保护性抗原的DNA序列设计引物,扩增保护性抗原基因。利用酶切、AT克隆方法快速分析筛选出保护性抗原基因片段的重组子,从而制备成芯片探针。DNA自动分析仪对克隆片段进行序列测定,生物信息学软件对其基因序列进行分析。结果根据炭疽杆菌保护性抗原设计的引物,可以扩增出长2 205 bp的保护性抗原基因,经酶切、AT克隆方法筛选出约7个长度不一的片段,对其片段进行序列测定并进行Blast序列比对得知这些片段均属于炭疽杆菌保护性抗原基因。结论利用PCR扩增产物并结合酶切、AT克隆方法可以快速、简便地制备基因芯片探针。     

炭疽杆菌保护性抗原基因芯片探针的制备

目的 快速制备炭疽杆菌保护性抗原基因芯片探针。方法 根据PubMed上公布的炭疽杆菌保护性抗原的DNA序列设计引物，扩增保护性抗原基因。利用酶切、AT克隆方法快速分析筛选出保护性抗原基因片段的重组子，从而制备成芯片探针。DNA自动分析仪对克隆片段进行序列测定，生物信息学软件对其基因序列进行分析。结果根据炭疽杆菌保护性抗原设计的引物，可以扩增出长2205bp的保护性抗原基因，经酶切、AT克隆方法筛选出约7个长度不一的片段，对其片段进行序列测定并进行Blast序列比对得知这些片段均属于炭疽杆菌保护性抗原基因。结论 利用PCR扩增产物并结合酶切、AT克隆方法可以快速、简便地制备基因芯片探针。     

Beta-galactosidase gene from Lactobacillus delbrueckii subsp. bulgaricus gets non-fusion expression in Escherichia coli

OBJECTIVE: To construct the food-grade recombinant probiotic strain with high activity beta-galactosidase, the beta-galactosidase gene (lacZ)from Lactobacillus delbrueckii subsp. bulgaricus was in non-fusion expressed in Escherichia coli. METHODS: From Lb. delbrueckii subsp. bulgaricus lacZ gene, the DNA sequence containing Shine-Dalgarno (SD) and ATGA sequences between upstream 18 bp and downstream 1 bp at start codon ATG was selected as upstream primer for PCR amplifying lacZ gene. Then lacZ cDNA was inserted into expression plasmid pMG36e to construct recombinant expression plasmid. Recombinant plasmids were introduced into E. coli, and positive clones were screened. To identify the gene recombination, the recombinant plasmid was cut by restriction enzyme and sequenced. To identify the protein expression, the beta-galactosidase activities of recombinant strains were determined. RESULTS: The restriction maps of recombinant plasmids were acceptable. The gene inserted into the recombinant plasmid had more than 99% homology with the lacZ gene of Lb. delbrueckii subsp. bulgaricus. The enzyme activity and enzyme activity ratio of E. coli DH5 alpha carrying pMG36e-lacZ 1.1480 were 3.074 U/mL and 6.939 U/mg pro respectively. The enzyme activity and enzyme activity ratio of E. coli DH5a carrying pMG36e-lacZ wch9901 were 4.755 U/mL and 8.537 U/mg pro respectively. CONCLUSION: lacZ from Lb. delbrueckii subsp. bulgaricus have gotten non-fusion expression in E. coli. The SD and ATGA sequences we selected can introduce lacZ non-fusion expression in E. coli.     

保加利亚乳杆菌β-半乳糖苷酶基因的扩增及其影响因素

目的 通过扩增得到保加利亚乳杆菌编码高效β-半乳糖苷酶的基因,并探讨影响扩增的因素。方法 采用溶菌酶加冻融法提取细菌DNA;选取不同的模板浓度、退火温度分别进行扩增。结果 模板浓度60ng／μl、退火温度66℃是保加利亚乳杆菌β-乳糖苷酶基因的较优扩增条件。结论 优化了扩增保加利亚乳杆菌β-半乳糖苷酶所需的退火温度、模板浓度等条件,提高了扩增产物的产量。     

Antibiotic susceptibility of strains in Chinese medical probiotic products

Objective： To investigate the susceptibility of strains separated from probiotic products for medical purpose to 14 antimicrobial agents. Methods：The single aerobic strains were isolated from these products respectively and disc agar diffusion assay was proceeded to determine the susceptibility. Results： Probiotics tested in the study mostly showed multiresistant to the agents. Lactobacillus acidophilus LAP, LAB, Lactobacillus bulgaricus LBJ and Streptococcus therrnophilus STJ were resistant to vancomycin. Conclusion： Drug resistance exists in most of commercial probiotics. The evaluation and monitoring of safety of probiotic products for medical purpose should be paid great attention.     

Non-Fusion and Fusion Expression of β-Galactosidase from Lactobacillus bulgaricus in Lactococcus lactis

Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation pr     

BALB/c小鼠乳糖酶基因Lac的克隆及其cDNA探针的制备

目的 提取、克隆BALB／c小鼠乳糖酶基因Lac,了解其序列及所编码蛋白的属性,并制备Lac cDAN探针,为进一步研究乳糖不耐受奠定基础。方法 取4周龄BALB／c小鼠小肠组织,用Trizol试剂盒提取总RNA,经RT—PCR、梯度PCR获取Lac cDNA。测序鉴定后将序列提交NCBI GenBank,同时利用生物信息学,对其编码产物进行预测。探针的制备采用随机引物法以地高辛标记。结果 所获得的Lac cDNA编码区有912bp,编码303个氨基酸残基,在其二级结构中存在一个糖苷键水解酶基序,C-末端有一疏水螺旋区。标记后的探针经检测,灵敏度达到1pg／μl。结论 所获得的Lac基因经鉴定确为乳糖酶基因,由它编码的蛋白产物是乳糖酶。标记的探针灵敏度很高,可以用于原位杂交实验。     

Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1： A Homolog of Human Lactase-Phlorizin Hydrolase

Objective To study the mechanism of lactose intolerance （LI） by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse （δ）. Crene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase （LPH） in human, rat, and rabbit. A coding sequence （CDS） fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinforrnatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosyl hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA （GenBank accession No： AY751548） provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.     

不同年龄BALB/c小鼠乳糖酶及基因调控水平研究

目的 通过测定不同年龄段小鼠乳糖酶活性和乳糖酶mRNA水平,探讨乳糖酶变化趋势及其基因调节途径。方法 分别选取3、5、7、9周龄的BALB／c小鼠,测定其乳糖酶活性及其mRNA水平。乳糖酶活性测定采用Dahlqvist改良法,mRNA水平采用原位杂交,半定量分析。结果 小鼠3周龄时乳糖酶活性最高,与其他各组有显著差异;9周龄小鼠乳糖酶活性最低,与其他三组有显著差异。而乳糖酶mRNA水平无差异。结论 结果提示断乳前小鼠乳糖酶活性有较高水平,断乳后乳糖酶活性显著降低,说明BALB／c小鼠也存在成年乳糖不耐受问题。各组乳糖酶mRNA水平无统计学差异,提示乳糖酶基因的调控应该是发生在翻译和／或后翻译水平,而并非是在转录水平。     

酵母菌药用乳糖酶制备新方法

目的:建立一种简易的从酵母菌制备克鲁维酵母乳糖酶的方法.方法:在30L发酵罐中进行发酵,用对羟基苯甲酸酯进行细胞破壁,用超微过滤方法浓缩破壁液.结果:乳糖酶的收率可达到每毫升发酵液含11.4 ONPG单位.从酵终细胞中释放乳糖酶可达70%,每毫升牛奶中加入1.2 ONPG单位制备的液态乳糖酶可使牛奶中的乳糖水解率达到70%.     

乳酸杆菌对H.pylori SS1诱导SGC7901细胞NF-κB活化和分泌IL-8的调节作用

目的 研究体外嗜酸乳酸杆菌标准株(Lactobacillus acidophilus, ATCC4356)、保加利亚乳酸杆菌(L.bulgaricus,LB)在幽门螺杆菌悉尼株(H.pylori SS1)诱导SGC7901细胞分泌IL-8过程中对NF-κB活化的调控作用.方法 以SGC7901细胞作为H.pylori SS1株感染的体外细胞模型,再以ATCC4356(ATCC4356干预组)和LB(LB干预组)培养至指数生长期调不同浓度与细胞共育1 h后,用H.pylori刺激3 h,采用免疫组化法和ELISA法观察乳酸杆菌对幽门螺杆菌诱导SGC7901细胞NF-κB活化核表达及对IL-8分泌量的影响.结果 与空白对照组比较,H.pylori诱导的SGC7901细胞核NF-κBp65表达阳性率及IL-8分泌量升高,差异具有统计学意义(P＜0.01).与H.pylori组比较,ATCC4356干预组在浓度为3×106 cfu/mL、3×107cfu/mL、3×108 cfu/mL时能降低NF-κBp65表达阳性率(P＜0.01).与H.pylori组比较,ATCC4356干预组在浓度为3×106 cfu/mL、3×107 cfu/mL时能减少IL-8的分泌量(P＜0.01).与H.pylori组比较,LB干预组在浓度为3×106 cfu/mL～3×108 cfu/mL时能降低NF-κBp65表达阳性率(P＜0.05).与H.pylori组比较,LB干预组在浓度为3×107 cfu/mL、3×108 cfu/mL时能减少IL-8的分泌量(P＜0.05).但两种乳酸杆菌浓度过高时可能无上述抑制作用或相反的增加NF-κB p65表达阳性率及IL-8的分泌量.结论 在一定的浓度范围内两种乳酸杆菌均能抑制H.pylori诱导的炎症反应,但高于一定的浓度值则会加重炎症反应,提示乳酸杆菌作为重要的胃内常驻菌保持适当的浓度对于维持胃内微生态平衡稳定的重要性.     

人同源盒基因Nkx3.1启动子的克隆及活性测定

目的：克隆Nkx3．1基因5’上游1．06kb片段，构建pGL3-1．06kb载体，测定其启动子活性。方法：采用PCR方法从人基因组DNA中扩增Nkx3．1基因5’上游1．06kb片段并构建到荧光素酶报道基因pGL3-basic载体中，与内参照质粒pRL-Tk共转染前列腺癌LNCaP细胞，通过双荧光素酶活性检验测定其启动子活性。结果：PCR扩增的1．06kb片段经测序正确无误；pGL3-1．06kb转染LNCaP细胞48h后，双荧光素酶活性测定M1／M2=2．7，为pGL3-control活性的1．5倍，为pGL3-basic活性的50倍。结论：克隆的人Nkx3．1基因5’上游1．06kb片段具有较强的启动子活性。