中国莱姆病螺旋体PD91重组OspC的鉴定和抗原性检测

目的 重组中国莱姆病螺旋体PD91外膜蛋白C(OspC)并在大肠埃希菌中表达，用于早期莱姆病诊断的研究。方法 用聚合酶链反应(PCR)扩增出PD91外膜蛋白C基因，定向克隆到表达载体PET-llD，构建重组质粒PET-llD-ospC。用PCR、限制性内切酶分析及序列测定等方法鉴定重组质粒。用Western Blot检测其抗原性。结果 OspC基因被正确克隆到表达载体PET-llD中。序列测定结果证实与国外已报道的序列存在一定的差异。OspC具有强抗原性。结论 该项研究为国内莱姆病的早期特异性诊断的研究奠定了基础。     

伯氏疏螺旋体PD91外膜蛋白C克隆表达及序列分析

目的 构建伯氏疏螺旋体PD91菌株外膜蛋白C(OspC)的表达载体，克隆表达OspC，用于莱姆病的预防、诊断和致病机理上的研究。方法用PCR扩增PD91 ospC基因，定向克隆到表达载体PET-11D，构建重组质粒。采用酶切分析及序列测定等方法鉴定重组质粒的正确性。结果 ospC基因被正确克隆到表达载体PET-11D中。序列测定结果证实与已报道的ospC基因序列同源性介于62％～86％之间。结论我国PD91菌株的ospC编码基因与已报道菌株的ospC菌株在同源性上存在较大的差异。PET-11D—ospC重组质粒的成功构建为我国莱姆病的进一步研究莫定了基础。     

伯氏疏螺旋体广西分离株外膜蛋白A基因的克隆和序列分析

目的：探求广西伯氏疏螺旋体外膜蛋白A（OSPA）基因变异情况。方法：应用聚合酶链反应从3株广西莱姆病螺旋体GXLD－4，9，18及1株贵州分离株长14全基因组DNA中将OSPA基因调出，并克隆到pGEM-T Easy Vector载体上，构建重组质粒，测序后与国外其它分离株进行同源性比较。结果：4株莱姆病螺旋体均扩增出约774bp的OSPA基因，编码258个氨基酸。4者之间核苷酸同源性98．8％－99．9％，氨基酸同源性98％－99％，其中GXLD－4与长14同源性最大。与国外分离株（10MT，Ya501)的同源性均较高。结论：莱姆病螺旋体OSPA基因在广西3个分离株及贵州株长14间差异较小，但与国外分离株存在一定差异。     

小鼠抵抗素基因及其反义核酸真核表达体系的构建与鉴定

目的构建载有小鼠抵抗素(resistin)基因及其反义核酸的重组真核表达质粒,为下一步进行resistin生物功能研究打基础。方法用resistin基因mRNA编码区序列特异引物,从小鼠脂肪组织中,通过RT-PCR的方法合成resistin cDNA,T4DNA连接酶将resistin cDNA克隆于pGEM-T载体,经双酶切及测序鉴定克隆成功后再亚克隆于pcDNA3.1(+)或pcDNA3.1(-)真核表达载体,并测序鉴定。结果PCR产物长度与resistin cDNA理论长度363bp相符;重组pGEM-T被EcoRⅠ和XbaⅠ内切酶切为约3000bp和355bp两个片段,测序结果表明插入pGEM-T的DNA片段的核苷酸序列与小鼠resistin基因mRNA编码区序列完全一致。重组pcDNA3.1(+)和pcDNA3.1(-)测序结果表明插入的DNA片段分别与小鼠resistin基因mRNA编码区序列和反义resistin基因mRNA编码区核苷酸序列一致。结论成功克隆载有resistin基因和载有resistin基因反义核酸的重组真核表达质粒。     

腰果主要过敏原Anao2基因克隆及原核表达

目的 克隆腰果主要过敏原Anao2基因,并利用pMAL-c表达载体表达该蛋白.方法 提取腰果总RNA,设计特异性引物,反转录-聚合酶链反应(RT-PCR)克隆腰果Anao2基因,将其反转录基因连入pMD18Tsimple vector,提取质粒、双酶切、鉴定并测序;将测序正确的片段连入原核表达载体pMAL-c,将重组质粒转入BL21宿主表达菌中,丙基-β-D-硫代吡喃半乳糖苷诱导表达目的蛋白Anao2.结果 测序结果表明克隆腰果Anao2基因片段全长为1 332 bp,编码443个氨基酸,与GenBank中蛋白序列完全相同;对获得的重组蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定,目的蛋白大小与理论值相符.结论 成功克隆并表达了腰果过敏原Anao2.     

小鼠抵抗素基因及其反义核酸真核表达体系的构建与鉴定

目的构建载有小鼠抵抗素（resistin）基因及其反义核酸的重组真核表达质粒,为下一步进行resistin生物功能研究打基础。方法用resistin基因mRNA编码区序列特异引物,从小鼠脂肪组织中．通过RT—PCR的方法合成resistin cDNA,T4DNA连接酶将resistin cDNA克隆于pGEM-T载体,经双酶切及测序鉴定克隆成功后再亚克隆于pcDNA3．1^（＋）或pcDNA3．1^（-）真核表达载体,并测序鉴定。结果PCR产物长度与resistin cDNA理论长度363 bp相符;重组pGEM—T被EcoR Ⅰ和Xba Ⅰ内切酶切为约3000bp和355bp两个片段,测序结果表明插入pGEM-T的DNA片段的核苷酸序列与小鼠resistin基因mRNA编码区序列完全一致。重组pcDNA3．1^（＋）和pcDNA3．1^（-）测序结果表明插入的DNA片段分别与小鼠resistin基因mRNA编码区序列和反义resistin基因mRNA编码区核苷酸序列一致。结论成功克隆载有resistin基因和载有resistin基因反义核酸的重组真核表达质粒。     

我国滇西北山区家犬莱姆病螺旋体基因的检测及序列分析

目的了解滇西北山区莱姆病伯氏疏螺旋体的自然感染及基因分型情况。方法以山区农村家犬作为指示动物,使用真空采血管抽取家犬全血,应用巢式PCR扩增犬血中伯氏疏螺旋体5S~23S rRNA间隔区片段,然后对阳性片段进行测序,并将所测序列与GenBank中注册的基因序列进行比较分析。结果共检测贡山、福贡2个县3个乡镇山村63只家犬血液,其中发现9只犬为阳性,阳性率14.29%。基因序列分析结果显示,当地犬感染的伯氏疏螺旋体为Borrelia garinii。结论首次证实云南省西北部山区家犬中存在莱姆病Borrelia garinii型伯氏疏螺旋体感染。     

淋病奈瑟菌外膜蛋白PorB编码基因的克隆及序列分析

目的 获取淋病奈瑟菌外膜蛋白PorB编码基因(porB)并构建其重组表达质粒。方法 根据porB已知序列，设计合成一对引物，用PCR方法从淋病奈瑟菌基因组DNA中扩增出porB基因片段，克隆到原核表达质粒pQE30中，转化大肠埃希菌M15感受态细胞，经酶切和PCR鉴定，然后进行测序。结果 porB基因体外扩增产物大小约为911bp。重组质粒经双酶切和PCR鉴定表明为正确重组子，测序结果与已知序列基本吻合。结论 成功克隆了淋病奈瑟菌外膜蛋白PorB编码基因，为下一步PorB蛋白的功能研究和淋病奈瑟菌蛋白质疫苗的研制提供了基本的物质基础：     

巢式PCR法检测鼠肝脾标本伯氏疏螺旋体

目的应用巢式PCR方法检测鼠肝脾组织中伯氏疏螺旋体外膜蛋白A（ospa）基因,了解淳安县鼠中伯氏疏螺旋体感染情况。方法于2012年8月收集淳安县102份鼠肝脾标本,采用巢式PCR法检测伯氏疏螺旋体ospA基因特异片段,统计标本阳性率。结果在38只黑线姬鼠中检测到伯氏疏螺旋体ospA基因特异片段阳性标本3份,在13只黄毛鼠中检测到伯氏疏螺旋体ospA基因特异片段阳性标本3份,鼠伯氏疏螺旋体总阳性率为5．88％（6／102）。结论淳安县鼠中检测到伯氏疏螺旋体ospA基因片段,有引起蜱媒传染病莱姆病发生的潜在可能,应引起公共卫生部门的重视。     

广西凭祥地区莱姆病螺旋体检测和基因分型研究

目的 调查广西凭祥地区莱姆病感染情况和基因型别.方法 2011年7月从广西凭祥地区采集蜱、啮齿动物和野鸟标本,分别采取煮沸法和Qiagen试剂盒提取蜱、啮齿动物脾脏以及鸟脾脏中莱姆病螺旋体基因组DNA;采用巢式PCR扩增莱姆病螺旋体5S～23S rRNA基因间隔区;对PCR扩增产物进行测序,并将序列结果与GenBank中莱姆病螺旋体5S～23SrRNA基因问隔区序列比对分析.结果 从3份啮齿动物标本中检测到莱姆病螺旋体5S～23SrRNA基因间隔区片段,啮齿动物的感染率为5.66％(3/53).其中一个序列与GenBank中Borrelia valaisiana基因型莱姆病螺旋体(序列号:HM100125.1,AB091455.1,AB091454.1,AB091453.1)同源性为100％;蜱和鸟标本中未检测到莱姆病螺旋体.结论 广西凭祥地区啮齿动物中存在莱姆病螺旋体B.valaisiana基因型感染.     

Synthetic recombinant vaccine expressing influenza haemagglutinin epitope in Salmonella flagellin leads to partial protection in mice.

The influenza virus haemagglutinin epitope 91-108, which is a conserved amino acid sequence in all type A H3 strains, was expressed in Salmonella flagellin, to evaluate its potential as a vaccine. For that purpose, a synthetic oligonucleotide comprising 54 bases coding for the corresponding sequence was inserted into the plasmid pLS408 and transformed into Escherichia coli JM101. Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative 'live vaccine' aroA mutant of Salmonella dublin. Rabbits immunized either with the live recombinant S. dublin or with the flagellin isolated from it, showed significant levels of IgG response against the synthetic peptide 91-108 as well as against the intact A/Texas/77 influenza virus. Mice immunized with the same preparations developed influenza-specific IgG antibodies in the blood and secreted IgA antibodies in their lungs. Furthermore, these mice showed about 50% protection against challenge infection with the virus. The most successful results were achieved by intranasal immunization with the isolated recombinant flagellin, when employed without the aid of adjuvant.     

Cloning and expression of recombinant flagellar protein flaB from Borrelia burgdorferi

OBJECTIVE: Lyme borreliosis is an arthropod transmitted infection caused by some species of the Borrelia genus. Current diagnosis employs serological testing and detection of Borrelia-specific antibodies. Using recombinant Borrelia burgdorferi antigens may improve assay specificity and sensitivity. One of the immunodominant Borrelia antigens that elicit a strong and early immune response is FlaB, making it appropriate for recombinant protein based serological diagnostic tests. MATERIAL AND METHODS: Borrelia burgdorferi genomic DNA was isolated and used as a template for the amplification of the flaB gene. The gene was cloned in the expression vector pGEX-2T. RESULTS: The amplified flaB gene was cloned in the expression vector yielding a GST-FlaB fusion gene. The gene ligated in-frame was expressed as the recombinant GST-FlaB protein. After visualization by polyacrylamide gel electrophoresis the successful expression of the FlaB protein was confirmed by immunoblotting. CONCLUSION: The expression and purification of the recombinant FlaB protein is a prerequisite for obtaining large amounts of the product through a simple and labour-free procedure, which will facilitate the diagnosis of Lyme disease.     

问号状钩端螺旋体外膜蛋白Loa22的克隆、表达及对豚鼠的免疫保护作用研究

目的构建问号状钩体强毒赖型56601株外膜蛋白Loa22的重组质粒,表达Loa22蛋白,研究该蛋白对豚鼠的保护作用。方法以问号状赖型钩体56601株基因组为模板扩增目的基因,将Loa22基因克隆至原核表达载体PQE-31,构建PQE31-Loa22重组体,将其转入大肠杆菌M15中诱导表达目的蛋白。于0、2、4周将蛋白及对照PBS分别经豚鼠腹股沟、腋下免疫豚鼠。每次剂量为蛋白50μg/只,末次免疫后2周,用培养3d的56601株钩体从腹腔攻击各免疫组豚鼠(1ml/只)。连续观察15d,观察各免疫组豚鼠发病情况,并取各免疫组豚鼠的肺、肝、肾做病理切片。分析蛋白Loa22对豚鼠的免疫保护作用。结果成功扩增出Loa22基因,构建原核重组质粒PQE31-Loa22。重组质粒能在大肠杆菌M15中高效表达Loa22蛋白。用Loa22蛋白主动免疫的豚鼠用56601株钩体攻击,结果显示无发病现象,而免疫对照组的豚鼠中出现了食欲不振、活动减少等现象,病理切片有明显改变。结论成功构建了Loa22重组原核表达质粒,表达纯化的蛋白可以使豚鼠抵抗同型钩体的攻击,免疫保护作用为82%。     

Sequence analysis of functional Apisimin-2 cDNA from royal jelly of Chinese honeybee and its expression in Escherichia coli

Apisimin is one of the functional peptides from royal jelly. The aim of this study was to analyze and in vitro express a new gene encoding Acc-apisimin-2 from Chinese honeybee (Apis cerana cerana) in Escherichia coli. Ninety-six clones containing apisimin expressed sequence tag (EST) were identified from 8568 effective ESTs of the cDNA library of Chinese honeybee worker heads. The coding region of the matured peptide from one clone containing Acc-apisimin-2 gene was sub-cloned into the prokaryotic expression vector pGEX-4T-2. The recombinant vector then was transformed into E. coli BL21 (DE3) for expression. The expression product was analyzed with SDS-PAGE and Western blot. The total length of the Acc-apisimin-2 cDNA was 379 bp, containing an open-reading frame (ORF) of 237 nucleotides encoding a 78 amino acid residue precursor. The Acc-apisimin-2 gene shared 100% homologies with Am-apisimin from A. mellifera, but 93% and 91% homologies with Aciapisimin from A. cerana indica and the previously reported Acc-apisimin-1 sequence (AY278991) on a nucleotide level, respectively. The GST-Acc-apisimin-2 fusion protein expressed in the recombinant vector was about 31 kDa in size and accumulated up to about 22.1% of the total bacterial proteins. About 50% of the recombinant protein was soluble. The fusion protein purified through affinity chromatography was cross reactive with GST antibody, which confirmed the successful expression of GST-Acc-apisimin-2.     

弓形虫微线体蛋白1部分基因的克隆、测序及表达

目的 构建弓形虫ZS2株pWR450-1－微线体蛋白1（MIC1）原核表达重组质粒,对MICI基因进行序列测定,并在不同大肠杆菌中表达。方法 用PCR技术从弓形虫ZS2株的基因组DNA中扩增编码MICI的基因片段,酶切,连接,重组入pWR450-1表达载体,再经含氨苄培养基筛选、酶切、PCR鉴定,进行序列测序后转化大肠杆菌TG－1、JM109（DE3）和DH5α。在不同菌体浓度及不同剂量异丙基－β－D－硫代半乳糖诱导下,用SDS－聚丙烯酰胺凝胶电泳鉴定MIC1融合蛋白的表达。结果 从ZS2株基因组DNA中扩增出特异的MIC1基因片段,克隆后获得pWR450-1/MIC1重组质粒,测序结果表明,MIC1这部分基因与弓形虫RH株相应基因序列完全一致,高度保守。该基因可在不同大肠杆菌中表达相对分子质量约70000的融合蛋白。结论 构建了弓形虫ZS2株pW450-1-MCI1重组质粒,为研究MIC1的结构与功能奠定了基础。     

解脲脲支原体的N端保守部分MB优化基因在大肠埃希菌中的高效表达

目的 制备N-端保守部分多条带抗原(MB),克隆并在大肠埃希菌中表达其基因,并鉴定重组MB的抗原性.方法分析14个不同血清型解脲脲支原体(Uu)的基因排列,根据大肠埃希菌偏爱密码子,优化、设计合成DNA序列编码的氨基酸保守序列,合成的基因克隆到表达质粒,重组蛋白诱导和亲和层析纯化.结果 通过对Uu 14个血清型MB抗原氮端的氨基酸序列的分析、比对,根据大肠埃希菌的偏好密码子优化、设计并合成碱基序列,目的基因片段与pET-41a构建重组质粒并转化大肠埃希菌后,IPTG诱导表达,SDS-PAGE分析,得到了分子量约为45 kd的重组蛋白,纯化后的MB抗原蛋白能够较特异地与特异性抗体结合,敏感性为85.7％,特异性为87.8％.结论 Uu N-端保守部分的MB蛋白已成功表达、纯化并被证明具有良好的免疫原性.     

Antigenic and genomic analysis of a Borrelia burgdorferi sensu stricto strain isolated from Ixodes ricinus ticks in Alto Adige-South Tyrol, Italy

A Borrelia burgdorferi sensu lato strain isolated from Ixodes ricinus ticks in Alto Adige-South Tyrol (Northern Italy) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins, Western immunoblotting analysis (WBA) with polyclonal and monoclonal antibodies, and pulsed-field gel electrophoresis (PFGE). The isolate named BZ6 was identified as belonging to the genospecies B. burgdorferi sensu stricto on the basis of its protein profile and its reactivity with monoclonal and polyclonal antibodies. The PFGE study performed with the two rare-cutting restriction enzymes MluI and SmaI confirmed the SDS-PAGE and WBA characterizations, but showed a genetic diversity between the isolate and two out of the three B. burgdorferi sensu stricto strains used in this study as controls, the American type strain B31 and the locally isolated strain BZ1. No difference in the PFGE patterns between the isolate BZ6 and the Swiss strain IRS was noted. Our findings show the value of PFGE analysis for classifying B. burgdorferi sensu lato isolates and for revealing their genetic diversity, and its usefulness for epidemiological investigations.     

Modification of the multiplex plasmid PCR assay for Borrelia miyamotoi strain LB-2001 based on the complete genome sequence reflecting genomic rearrangements differing from strain CT13–2396

The genome of Borrelia spp. consists of an approximate 1 megabase chromosome and multiple linear and circular plasmids. We previously described a multiplex PCR assay to detect plasmids in the North American Borrelia miyamotoi strains LB-2001 and CT13–2396. The primer pair sets specific for each plasmid were derived from the genome sequence for B. miyamotoi strain CT13–2396, because the LB-2001 complete sequence had not been generated. The recent completion of the LB-2001 genome sequence revealed a distinct number of plasmids (n = 12) that differed from CT13–2396 (n = 14). Notable was a 97-kilobase plasmid in LB-2001, not present in CT13–2396, that appeared to be a rearrangement of the circular plasmids of strain CT13–2396. Strain LB-2001 contained two plasmids, cp30–2 and cp24, that were not annotated for strain CT13–2396. Therefore, we re-evaluated the original CT13–2396-derived multiplex PCR primer pairs and determined their location in the LB-2001 plasmids. We modified the original multiplex plasmid PCR assay for strain LB-2001 to include cp30–2 and cp24. We also determined which LB-2001 plasmids corresponded to the amplicons generated from the original CT13–2396 primer sets. These observations provide a more precise plasmid profile based on the multiplex PCR assay and reflect the complexity of gene rearrangements that occur in B. miyamotoi strains isolated from the same geographic region.