牙龈卟啉单胞菌菌毛融合抗原基因果实特异表达载体的构建及意义

目的构建含牙龈卟啉单胞菌菌毛融合抗原基因的番茄果实特异表达载体,并提高抗原基因的表达量和免疫原性,为其在番茄果实内表达和研制有效的转基因植物牙周炎疫苗奠定基础。方法通过PCR获得番茄果实特异表达启动子E8的核心序列片段（约1．11kb）,同时合成通过柔性接头连接的霍乱毒素B亚基（Cholera Toxin B Subunit,CT8）和牙龈卟啉单胞菌菌毛FimA（266-337）（约600bp）序列的基因片段,利用重叠区扩增基因拼接法将两基因片段连接,得到重组片段E8．CTB．FimA（266-337）,双酶切E8-CTB-FimA（266-337）片段和植物表达载体pB1121,酶切产物连接后,获得重组载体pBIE8-CTB-FimA（266-337）,利用酶切方法和测序方法对pBIE8-CTB-FimA（266-337）进行鉴定。结果基因测序及酶切结果表明特异表达载体已成功构建。结论本研究成功构建了含牙龈卟啉单胞菌菌毛融合抗原基因的番茄果实特异表达载体;该载体可用于转基因植物牙周炎疫苗的研制。     

牙龈卟啉单胞菌脂多糖对人主动脉内皮细胞IL-10和MCP-1表达的影响

目的探讨牙龈卟啉单胞菌脂多糖(LPS)对人主动脉内皮细胞白细胞介素(IL)-10和单核细胞趋化蛋白(MCP)-1表达的影响。方法用不同浓度(0、10、100、150 ng/ml)的牙龈卟啉单胞菌脂多糖分别作用于人主动脉内皮细胞。酶联免疫吸附法、聚合酶链反应检测IL-10和MCP-1的表达。结果在一定浓度范围内,牙龈卟啉单胞菌LPS呈剂量依赖性促进人主动脉内皮细胞(HAECs)MCP-1的表达,同时减低IL-10的表达水平。结论牙龈卟啉单胞菌LPS可诱导人主动脉内皮细胞IL-10和MCP-1表达紊乱,从而促进动脉粥样硬化的发生。     

牙龈卟啉单胞菌与口腔种植体周围重建骨的相关性研究

目的 探讨牙龈卟啉单胞菌(Porph yromonas gingivalis,P.g)在种植体周围骨重建中的意义.方法 收集40例口腔种植骨重建患者的菌斑,同时进行口腔卫生和牙周维护治疗调查,菌斑检测、PCR扩增检测口腔的P.g以及种植体周边缘骨吸收测定.结果 共收集40例198份菌斑,骨吸收＜0.3 mm,0.3～0.6 mm及＞0.6 mm组,其P.g附着率分别为28.6％,45.3％及67.2％,经统计学检验,差异有统计学意义(P＜0.05).牙周末维护有利于菌斑堆积,其P.g检出率高.结论 P.g可能影响种植体周围骨重建,是导致边缘骨吸收的原因之一.     

16S rDNA多重PCR检测牙龈卟啉单胞菌、伴放线放线杆菌和齿垢密螺旋体及混合感染与慢性牙周炎病变程度关系(英文)

目的 建立多重16S rDNA PCR方法用以同时检测慢性牙周炎(CP)临床标本中牙龈卟啉单胞菌(Pg)、伴放线放线杆菌(Aa)和齿垢密螺旋体(Td),并了解不同感染情况与慢性牙周炎严重程度的关系。方法 采集152例CP患者牙周袋标本,并按Hugoson的方法将其分为轻、中和重度三类,另采集30例牙周健康者龈沟标本作为正常对照。上述临床标本置于200μl裂解缓冲液中,100℃冰浴10min后取10μl上清液直接作为PCR的模板。采用常规酚-氯仿法提取Pg ATCC33277株、Aa Y4株、Td FM株和E.coli DH5α株的DNA分别作为PCR的阳性和阴性对照。采用Pg、Aa和Td 16S rDNA特异性引物,建立多重PCR检测上述标本。3例Pg、Aa和TdPCR结果均阳性患者牙周袋标本目的扩增片段T-A克隆后测序。采用X2检验分析Pg、Aa和/或Td感染率与牙周炎严重程度之间的关系。结果 所建立的多重16S rDNA PCR最低可检出10个Pg、20个Aa和20个Td细胞。Pg、Aa和Td 16S rDNA扩增片段测序结果与已报道的相应序列比较,相似性分别高达99.45％、97.08％和96.59％。30例牙周健康者龈沟标本中,仅有1例(3.3％)Pg、2例(6.7％)Aa的16S rDNA扩增结果阳性,其余标本均阴性。152例CP患者牙周袋标本中,147例(96。7％)检出Pg、Aa和/或Td的16S rDNA,5例(3.3％)扩增结果均阴性。Pg的PCR检测阳性     

甲型副伤寒沙门菌ompN基因原核表达系统构建及其产物免疫保护作用

目的了解甲型副伤寒沙门菌外膜蛋白ompN基因携带率及其重组表达产物抗原性和免疫保护作用。方法采用PCR扩增甲型副伤寒沙门菌参考标准株50001及126株临床菌株ompN基因并测序。构建ompN基因原核表达系统,Ni-NTA亲和层析法提取目的重组蛋白rOmpN。采用家兔免疫法和ELISA检测rOmpN免疫原性。微量肥达试验和激光共聚焦显微镜法分别检测OmpN在甲型副伤寒沙门菌株中表达率及其膜定位。采用小鼠感染模型了解rOmpN对甲型副伤寒沙门菌致死性感染的免疫保护作用。结果所有甲型副伤寒沙门菌株中均扩增出全长ompN基因片段,其核苷酸和氨基酸序列相似性分别高达98.6%~99.9%和98.7%~100%。OmpN是甲型副伤寒沙门菌跨膜蛋白。ompN基因表达系统能表达rOmpN,免疫家兔后能产生高效价抗血清。98.2%(55/56)甲型副伤寒病人血清标本中rOmpN-IgG阳性,rOmpN兔抗血清能有效凝集甲型副伤寒沙门菌。100和200 μg rOmpN对感染小鼠的免疫保护率分别为60.0%(9/15)和73.3%(11/15)。结论甲型副伤寒沙门菌ompN基因分布广泛且序列保守, rOmpN有较强的抗原性和免疫保护作用。     

牙龈卟啉单胞菌对血管内膜粘附因子和金属蛋白酶的影响(英文)

目的:观察牙龈卟啉单胞菌(Pg,为牙周病的主要致病菌)对血管内膜粘附因子和金属蛋白酶的影响。方法:大鼠120只,按数字表随机分为空白对照组、Pg低剂量组(2ml)与Pg高剂量组(5ml),各40只,后两组每3d分别接受相应剂量Pg肌注,且均不进行抗生素干预,历时12周。取其静脉血,检测三组血液中的细胞间粘附分子-1(ICAM-1)、血管细胞粘附分子-1(VCAM-1)、基质金属蛋白酶(MMP)-3及MMP-9的水平。结果:与空白对照组相比,Pg低剂量组与高剂量组大鼠静脉血中ICAM-1[(175.79±14.30)ng/ml比(182.62±15.07)ng/ml、(189.39±14.93)ng/ml]、VCAM-1[(256.49±37.17)ng/ml比(271.58±32.85)ng/ml、(286.66±30.66)ng/ml]、MMP-3[(3.23±0.69)ng/ml比(3.61±0.82)ng/ml、(3.97±0.83)ng/ml]及MMP-9[(1.30±0.39)mg/L比(1.48±0.39)mg/L、(1.67±0.45)mg/L]含量显著增加(P0.05或0.01),且与Pg低剂量组相比,Pg高剂量组上述因子含量显著增加(P0.05)。结论:牙龈卟啉单胞菌可引起大鼠血清中血管内膜粘附因子和金属蛋白酶含量的显著增加,可加剧冠心病的病理演变。     

Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8％ of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1immunogenicity and antigenicity.     

Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

AIM: To insert the constructed TGF-beta(1) epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-beta(1) antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti-TGF-beta(1) vaccine. METHODS: The TGF-beta(1) encoding epitope gene (the mature TGF-beta(1) from 78-109 amino acid residues, TGF-beta(1)(32)) was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-beta(1)(32) vector. The HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-beta(1)(32) and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T(4) ligase. The fusion gene fragments HBcAg1-71-TGF-beta(1)(32)- HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification with Ni(+2)-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS: Enzyme digestion analysis and sequencing showed that TGF-beta(1) epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass (Mr) of the expressed product by pET28a (+)/CTC was Mr 24,600. The output of the target recombinant protein was approximately 34.8% of the total bacterial protein, mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-beta(1) polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-beta(1) antigenicity and could be used as anti-TGF-beta(1) vaccine. CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF-beta(1) epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-beta(1). The expressed TGF-beta(1) epitope gene shows good immunogenicity and antigenicity.     

葡萄球菌肠毒素A基因真核表达系统的构建及其目的重组蛋白的鉴定

目的 克隆金黄色葡萄球菌肠毒素A(SEA)基因、构建其真核表达系统及目的重组蛋白的表达情况。方法 采用高保真PCR从金黄色葡萄球菌ATCCl3565菌株中扩增全长SEA，目的扩增片段T-A克隆后测序。经核酸内切酶酶切重组质粒pUCm-T-SEA获得的pUCm-T-SEA基因片段与真核表达载体pPIC9K连接。采用电融合法将重组真核载体pPIC9K-SEA转化入P．pastoris GSll5株，构建成真核表达系统pPIC9K-SEA-P．pastoris GSll5。采用含G418抑制剂的YPD琼脂平板和PCR筛选并鉴定pPIC9K-SEA-P．pastoris GSll5。0．5％(V：V)甲醇诱导下，采用SDS-PAGE检查BMMY液体培养基上清中重组SEA(rSEA)的表达情况。结果可从S．aureus ATCCl3565株DNA中扩增获得SEA目的片段。与报道的SEA基因序列(GenBankNo．：AP004828 and L22566)比较，所克隆的SEA基因核苷酸及氨基酸序列的相似性分别高达98．84％～100％和100％。在甲醇的诱导下，pPIC9K-SEA-P．pastoris GSll5能表达在SDS-PAGE图谱位于预期位置的rSEA。结论我们成功地构建了能表达SEA的真核表达系统，为进一步分析SEA分子中毒性相关活性位点、定位突变获得减毒或无毒的突变体奠定了基础。     

人护骨素-谷胱甘肽转硫酶融合蛋白基因的构建与表达

目的 克隆人护骨素(OPG)成熟肽段编码区基因并研究在大肠杆菌中OPG-谷胱甘肽转硫酶(GST)的融合蛋白的表达。方法 采用RT-PCR从人骨肉瘤细胞系MG63的总RNA中扩增OPG成熟肽段编码区cDNA，构建原核表达载体pGEX-4T-1，转化大肠杆菌后经0．1mmoL／L异丙基硫代半乳糖苷(IPTG)诱导后收集菌体蛋白，以SDS—PAGE电泳及Western印迹鉴定蛋白表达，应用谷胱甘肽-Sepharose4B柱亲和层析纯化目的蛋白。结果 获得人OPG成熟肽段编码区cDNA，转化菌株可表达人OPG-GST融合蛋白，蛋白表达量约为菌体总蛋白的15．5％。Western印迹表明在相对分子质量66000处融合蛋白能与人OPG多克隆抗体特异性结合。经亲和层析后获得纯度为95．7％的OPG—GST融合蛋白。结论 获得人OPG成熟肽段全长cDNA，在大肠杆菌中表达OPG—GST融合蛋白并以亲和层析法进行纯化。     

隐孢子虫Gal／GalNAc特异性凝集素p30原核表达系统的构建及鉴定

目的获取隐孢子虫Gal／GalNAc特异性凝集素p30基因,并构建原核表达系统,获得相应的重组蛋白。方法采用聚合酶链反应（PCR）技术,从微小隐孢子虫基因组中扩增出p30基因,然后将其克隆入pMD18-T载体,转化后挑取阳性克隆进行酶切和测序鉴定,并进行生物信息学分析及预测。再将亚克隆与表达载体PET-28a（＋）分别酶切并连接,经IPTG诱导表达,表达产物用Ni—NTA亲和层析法纯化,SDS—PAGE和Western blotting检测表达效果。结果PCR扩增得到特异的微小隐孢子虫Gal／GalNAc特异性凝集素p30基因,测得的核苷酸序列及其推导的氨基酸序列与GenBank上提交的序列同源性分别为98％～100％和99％～100％,理论等电点和分子量分别为6．4854和31842Da,并存在9个潜在的抗原表位。经酶切、测序结果表明质粒已导入Rosetta。SDS—PAGE和Western blotting证实重组菌成功表达融合蛋白。结论成功构建微小隐孢子虫Gal／GalNAc特异性凝集素p30原核表达系统。     

肝片吸虫谷胱甘肽S-转移酶基因的克隆表达及活性分析

目的克隆和表达肝片吸虫谷胱甘肽S-转移酶基因(FhGST)以研究重组蛋白免疫学特性。方法参考Gen-Bank上发表的FhGST基因序列设计一对特异性引物,利用RT-PCR法扩增FhGST基因,将扩增产物克隆入pET-30a(+),构建重组表达载体。经PCR、双酶切和序列测定鉴定后转入E.coliBL21(DE3)宿主菌,经IPTG诱导表达,SDS-PAGE和Western blotting对该重组蛋白进行分析和鉴定。结果成功获得肝片吸虫GST基因全长为657bp的编码序列,SDS-PAGE结果表明重组蛋白分子量为31.3kDa,以包涵体形式表达。Western blotting结果证实重组蛋白能被其免疫的SD大鼠血清识别,表明其具有免疫原性;并且能与感染肝片吸虫的绒山羊血清发生反应,表明重组蛋白具有免疫反应性。结论成功的克隆了肝片吸虫谷胱甘肽S-转移酶基因,实现了在原核表达系统中高效表达重组蛋白,并具有良好的免疫学活性。     

丙型肝炎病毒非结构蛋白NS5A反式激活基因4剪切体Ns5atp4a基因原核表达载体的构建和表达

目的构建丙型肝炎病毒非结构蛋白NS5A反式激活基因4剪切体（NS5ATP4A）的原核表达载体并进行表达。方法用PCR扩增NS5ATP4A基因,克隆入pET32a（＋）质粒,构建pET32a（＋）-NS5ATP4A表达重组体,转化DH5α和BL21菌,经IPTG诱导。SDS-PAGE分析,Western Blot验证蛋白带。结果成功构建大肠杆菌原核表达载体。经IPTG诱导,得到了目的蛋白分子量为35kD,用Western Blot证实其具有良好的抗原性。结论构建原核表达载体pET32a（＋）-NS5ATP4A,为抗原的制备及临床检测奠定了基础。