原位整合型枯草芽孢杆菌芽孢表面展示载体的构建

为证明原位整合能否用于枯草芽孢杆菌芽孢表面展示外源蛋白,本研究将芽孢衣壳蛋白CotB、CotZ的编码序列(不含终止密码子),与绿色荧光蛋白基因重组,构建含有融合片段cotB-gfp和cotZ-gfp的原位整合型表面展示载体pEYW25、pEYW26;将上述质粒分别转入枯草芽孢杆菌(Bacillus subtilis PY79)获得重组菌株BSYW25和BSYW26。荧光显微镜下观察上述重组菌的芽孢,只有BSYW26能检测到荧光,Western blot结果显示BSYW26的芽孢外壳蛋白中含有GFP。结果表明原位整合作为一种新的表面展示方法能够应用于枯草芽孢杆菌芽孢表面展示。     

猪链球菌2型表面蛋白sp融合基因的构建与高效表达

利用PCR方法从猪链球菌2型四川资阳分离株449-1扩增出sp基因,克隆到pMD18-T载体中,构建出克隆载体pMD18T-sp。以SalⅠ和BamHⅠ从pMD18T-sp中切下目的sp基因片段并克隆到质粒pMAL-p2X中,构建重组表达质粒pMALp2X-sp,转化宿主菌TB1中进行诱导表达。分析表明sp基因序列比GenBank报道序列AY864331少了270 bp;SDS-PAGE电泳检测结果表明,重组菌株表达出了136 Ku左右的目的蛋白MBP-Sao,目的蛋白为可溶表达,约占菌体蛋白总量的12.3%。表达的蛋白可用Amylose树脂纯化。将纯化的融合蛋白MBP-Sao免疫家兔,所得抗血清经ELISA检测,结果显示融合蛋白MBP-Sao包板抗体效价达1:16 000,且与猪链球菌2型和9型呈阳性反应,而与沙门氏杆菌及巴氏杆菌呈阴性反应。本试验对猪链球菌2型表面蛋白Sao的高效表达及其免疫原性进行了初步研究,为进一步研究猪链球菌2型表面蛋白Sao的结构和功能奠定了基础。     

猪链球菌2型38 000蛋白主要功能区基因的克隆与表达

根据GenBank中已发表的猪链球菌2型38000蛋白基因核苷酸序列,设计合成1对特异性引物,采用PCR方法,以四川分离株猪链球菌2型基因组DNA为模板扩增38000蛋白主要功能区基因。将PCR产物纯化后与pMD18-T连接转化宿主菌DH5α,提取阳性质粒,进行PCR和酶切鉴定。将目的片段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),于37℃、0.8mmol/LIPTG条件下进行诱导表达,结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子质量约为35000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猪链球菌2型阳性血清发生特异性反应。     

牛分枝杆菌mpb64和Ag85B融合基因在大肠埃希氏菌中的原核表达

以牛分枝杆菌Vallee株基因组DNA为模板,应用PCR扩增获得mpb64和Ag85B两个目的基因片段,采用重叠延伸剪接技术（SOE）剪接mpb64和Ag85B,得到融合基因mpb64-Ag85B;将融合基因片段先克隆于pMD 18-T载体,再亚克隆到表达载体pET32a（＋）中,得到重组质粒pET64-85.该重组质粒经核苷酸序列测定,显示其中的外源片段与期望的序列一致;其BL21（DE3）转化菌经IPTG诱导表达带有6个组氨酸标签的融合蛋白;用Ni2＋螯合层析方法纯化融合蛋白,Western-blotting分析结果显示,该融合蛋白能与抗牛分枝杆菌阳性血清发生反应.     

PCV2衣壳蛋白羧基端基因在T4噬菌体表面的展示及ELISA方法的建立

用PCR扩增猪圆环病毒Ⅱ型广东分离株的衣壳蛋白羧基端基因,将PCR产物连接到pR质粒,转化DH5α细胞,筛选阳性克隆进行PCR鉴定并测序后,转化E2菌,将重组E2菌与缺陷型噬菌体T4-Z1同源重组后,得到重组噬菌体,经SDS-PAGE和Western blotting分析,表明衣壳蛋白片段在噬菌体表面正确展示,表达的融...     

大鼠成熟的肺表面活性蛋白B的原核表达及纯化

为表达和纯化SP-B蛋白,先对SP-B基因进行稀有密码子优化,PCR反应获得SP-B片段,构建重组质粒pGEX4T-1/SP-B,转化到E.coli BL21(DE3)中诱导表达;用SDS-PAGE与Western blot进行检测;用GSTPrep FF 16/10对融合蛋白进行纯化。经双酶切鉴定证实质粒中插入基因长250bp,测序结果与大鼠SP-B cDNA序列相符;质粒转化后用IPTG进行诱导,在分子质量约34ku处出现1个新条带,与pGEX4T-1/SP-B蛋白预期大小一致,且该融合蛋白能可溶性表达并被纯化。结果表明成功构建了pGEX4T-1/SP-B重组质粒,表达、纯化得到GST/SP-B蛋白,为研究肺表面活性物质替代药物奠定了基础。     

布鲁菌外膜蛋白OMP25的原核表达

为了克隆布鲁菌外膜蛋白OMP25基因并构建基因的原核表达系统,试验采用聚合酶链反应(PCR)扩增得到布鲁菌基因组OMP25基因片段,T-A克隆后测定核苷酸序列,将目的基因定向插入原核表达载体pET-32a,经双酶切和DNA测序,再将构建的重组质粒转化到E.coil DH5α、Rosetta,经IPTG诱导后用SDS-P...     

牛分支杆菌MPB70和CFP10-ESAT6基因的表达与检测

以牛分支杆菌染色体DNA为模板,分别以MPB70、CFP10-ESAT6融合蛋白基因特异性引物进行PCR扩增,获得约600 bp的DNA片段,并将其克隆入PQE30质粒,构建出原核表达载体PQE30-MPB70,PQE30-CFP10-ESAT6.将质粒转化至感受态DH5α中,经IPTG诱导和SDS-PAGE分析,可见相应外源蛋白带.纯化蛋白后作为包被抗原建立ELISA方法,为进一步研究牛结核病诊断方法奠定基础.     

猪链球菌2型mrp基因ORF1序列的克隆及原核表达

根据猪链球菌2型溶菌酶释放蛋白基因（mrp）的序列,设计并合成了1对特异性引物,以青海株的基因组DNA为模板扩增了mrp基因ORF1序列。将PCR产物进行了T/A克隆,转化大肠杆菌,鉴定成功获得目的片段后,将其定向亚克隆到pET-28a（＋）中,构建了原核表达质粒pET-28a-mrp,并将其转化至大肠杆菌BL21感受态细胞中,经1 mmol/L IPTG诱导和SDS-PAGE分析,出现了与预期目的蛋白一致的外源蛋白带（27.0 ku）。Western-blot分析表明,该融合蛋白具有MRP的抗原表位。研究结果为今后开展该病的免疫学研究奠定了一定基础。     

利用MS2识别序列标签纯化技术富集猪链球菌小RNA结合蛋白

为建立理想的猪链球菌RNA结合蛋白筛选方法,以该菌小RNA rss04和rss06为研究对象,通过纯化MS2序列识别蛋白MS2-MBP,构建表达MS2融合RNA(MS2-rss04与MS2-rss06)的质粒p SET2-MS2-rss04、pSET2-MS2-rss06及阴性对照质粒p SET2-MS2-negative,将上述质粒分别电转化至猪链球菌小RNA缺失株Δrss04、Δrss06及野生株P1/7,通过MS2识别序列标签纯化,收集洗脱液,提取RNA。结果显示:表达MS2融合RNA的样品,rss04和rss06分别富集16倍和110倍,而阴性对照组未检测到rss04和rss06的富集。结果表明:MS2识别序列纯化体系成功建立,可用于猪链球菌RNA结合蛋白的筛选,为深入研究猪链球菌小RNA功能提供技术平台。     

Identification and distribution of putative virulent genes in strains of Streptococcus suis serotype 2

In order to identify gene sequences unique to the virulent strains, suppression subtractive hybridization (SSH) was conducted using virulent Streptococcus suis type 2 (SS2) strain HA9801 and avirulent S. suis type 2 strain T15. Thirty genomic regions were absent in T15, and the DNA sequences of these regions in HA9801 were determined. These DNA fragments, containing putative virulence genes, encoded 28 proteins that were homologous to proteins involved in various aspects of cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems and others of unknown function. According to the published SS2 genomic sequence of the Chinese strain 98HAH33, PCR primers for 14 significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis strains from different sources, serotypes, regions, groups and times. The results showed that these 14 DNA fragments were widely distributed in 37 detected SS2 strains, yet were absent among the avirulent strain T15. Moreover, these fragments could be detected in other serotypes of S. suis, but each serotype had a different distribution of the fragments.     

新城疫病毒F基因的克隆表达及重组蛋白的反应原性分析

为分析新城疫病毒(NDV)融合蛋白(F)及其突变体(Fm)基因的反应原性,以携带有NDV-F和NDV-Fm基因的质粒为模板设计引物,PCR扩增产物经双酶切后分别连入原核表达载体pET-SUMO、pET-28a,构建重组质粒pET-SUMO-F、pET-28a-Fm,将重组质粒转化入宿主菌Rosetta 2感受态细胞,在IPTG诱导下表达。SDS-PAGE和Western blotting结果显示,NDV-F和NDV-Fm在原核系统中表达后分别获得了相对分子量为64.7 kD和48.7 kD的重组蛋白;重组蛋白能被抗NDV鸡阳性血清识别。试验表明,NDV-F和NDV-Fm可以在原核系统中表达,且具有良好的反应原性。     

Construction of the Shuttle Vector Expressing Green Fluorescent Protein and its Application in Lactobacillus acidophilus

In order to further study the mechanism of Lactobacillus acidophilus,GFP labeling shuttle expression vector pSET4s-P1-GFP-P2 was constructed by inserting green fluorescence protein (GFP),upstream and downstream homology arm of Lactobacillus acidophilus based on temperature shuttle expression vector pSET4s.Recombinant Lactobacillus acidophilus expressing green fluorescence gene was constructed by transforming pSET4s-P1-GFP-P2 into Lactobacillus acidophilus using electrotransformation.We could obtain the recombinant Lactobacillus acidophilus by resistance screening and temperature screening,and named it as ΔMG6243(GFP) which could expressed GFP gene stably by fluorescence microscopy observation and Western blotting analysis.We could analyse the genetic stability and biological characteristics with fluorescence microscopy observation and plate count.Green fluorescent of recombinant Lactobacillus acidophilus could be observed in the blue light,and also be observed after 10 generations.There were significant differences neither growth characteristics nor pH and salt tolerance compared with the wild mushrooms.The results showed that GFP labeling recombinant Lactobacillus acidophilus was constructed successfully.Exogenous gene could be non-resistant and integrative to express in the Lactobacillus acidophilus with the vector.It established the foundation for construction of recombinant Lactobacillus acidophilus.At the same time,the role of the GFP as marker laid the foundation for distribution,adhesion of probiotic in animal gastrointestinal tract and the mechanism of Lactobacillus acidophilus.     

新疆绵羊种布鲁氏菌HtrA基因与GroEL基因的克隆及其原核表达

根据已发表的牛流产型布鲁氏菌HtrA（High temperature requinnent A）基因、GroEL（热休克蛋白）基因设计特异性引物，从新疆绵羊种布鲁氏菌基因组中扩增出HtrA、GroEL基因片段，将HtrA、GroEL基因片段纯化后分别克隆到T载体上测序，结果表明新疆绵羊种布鲁氏菌HtrA基因片段长1542bp，编码513个氨基酸，与发表的牛种（B．abortus）、羊种（B．melitensis）、猪种（B．suis）的HtrA基因序列的同源性分别为99．68％、99．81％、99．55％。GroEL基因片段长1641bp，编码546个氨基酸，与B．melitensis、B．suis以及B．aborms GroEL基因的核苷酸序列同源性分别为99．88％、99．82％、99．88％。HtrA基因和GroEL基因与发表的B．abortus、B．melitensis、B．suis的HtrA基因和GroEL基因序列的具有很高的同源性。按正确的阅读框架分别将两基因片段定向克隆到表达载体pET．28a上，将重组质粒转化到大肠杆菌BL21菌株，经IPTG诱导表达，SDS-PAGE电泳和western blot分析表明，HtrA、GroEL基因能在大肠杆菌中成功表达，表达的蛋白分子量都约为60Ku，并能和布鲁氏菌免疫兔子产生的抗体发生特异性的结合。     

绿色荧光蛋白穿梭载体的构建及其在嗜酸乳杆菌中的应用

为深入研究嗜酸乳杆菌的作用机理,以温敏型穿梭载体pSET4s为基本骨架,将绿色荧光蛋白(GFP)和嗜酸乳杆菌上、下游同源臂插入该载体,构建GFP标记的穿梭表达载体pSET4s-P1-GFP-P2。利用电转化方法将穿梭表达载体pSET4s-P1-GFP-P2转化入嗜酸乳杆菌,通过抗性和温度双重筛选获得能稳定表达GFP的重组嗜酸乳杆菌,将Western blotting和荧光显微镜观察验证的重组菌命名为ΔMG6243(GFP)。利用荧光显微镜观察和平板计数的方法对重组菌进行遗传稳定性和生物学特性研究。结果显示,重组菌在蓝光激发下可见明显绿色荧光,连传10代仍可见明显荧光,与野生菌相比,其生长特性、酸碱盐耐受性均无显著差别。结果表明,本试验已成功构建稳定表达GFP基因的重组嗜酸乳杆菌,利用温敏型穿梭载体pSET4s实现了外源基因在嗜酸乳杆菌中非抗性、整合型表达,为嗜酸乳杆菌基因重组菌的构建奠定了基础,同时GFP的标记作用也为嗜酸乳杆菌在动物体内分布、定植规律和作用机理创造了可行的条件。     

猪链球菌Sao-M和IdeSsuis蛋白的原核表达与反应原性分析

通过原核表达系统表达猪链球菌Sao-M和Ide_(Ssuis)基因,并分析其反应原性。根据GenBank数据库发表的序列,利用分子生物学软件设计了2对特异性引物,通过PCR扩增Sao-M和Ide_(Ssuis)基因,经测序分析后,分别克隆到pET28a(+)和pMAL-c2X,构建重组表达质粒pET28a:Sao-M和p MAL-c2X:Ide_(Ssuis),然后将鉴定为阳性的重组质粒分别转入宿主菌BL21和Rosetta2中用IPTG进行诱导表达,通过免疫印迹进行鉴定。实验表明,两种重组蛋白在原核系统中获得了高效的表达,并能与相应的阳性抗血清发生特异性反应。本研究为进一步探究Sao-M和Ide_(Ssuis)蛋白生物学功能及其在猪链球菌致病机制上的作用提供了依据。     

Testing meningeal strains of Streptococcus suis to detect M protein genes.

Previous reports have suggested that the surface proteins found in meningeal strains of Streptococcus suis might be similar to the M protein of group A streptococci. Fifty-five strains of S suis, including human and swine meningeal and pneumonic isolates, were tested for M protein genes by DNA probes representing the constant domain of the 3' end of the group A, M protein gene. None of the S suis strains examined was positive, indicating that these organisms either lack M protein genes or harbour different genes, not expressing the constant domains of protein M from group A.     

细胞渗透肽TAT与3种弓形虫抗原的融合重组表达

为了探索新型弓形虫疫苗的传递系统,本试验分别构建了细胞渗透肽反式转录激活因子(TAT)与3种弓形虫抗原和增强型绿色荧光蛋白(EGFP)融合表达的重组质粒,即pET28a-TAT-EGFP,pET28a-TAT-SAG1,pET28a-TAT-GRA4和pET28a-TAT-AMA1质粒。重组质粒被转化到大肠杆菌中并通过IPTG诱导,成功进行了融合表达,表达产物采用Ni-NTA树脂进行纯化,然后进行SDS-PAGE分析。结果得到31 ku的TAT-EGFP融合蛋白、34 ku的TAT-SAG1融合蛋白、38 ku的TAT-GRA4融合蛋白和62 ku的TAT-AMA1融合蛋白,经过Western blotting分析,感染弓形虫的小鼠血清可特异性地识别融合TAT的SAG1、GRA4蛋白和微弱地识别TAT-AMA1蛋白。     

基于细菌表面展示技术的猪链球菌病-副猪嗜血杆菌病二联亚单位疫苗的小鼠保护效果评价

为评价猪链球菌病-副猪嗜血杆菌病表面展示二联亚单位疫苗对小鼠的保护效果,本研究在应用融合PCR构建副猪嗜血杆菌(HPS)保护性抗原AfuA-OppA2和CdtB-OppA基因的串联序列基础上,将其分别插入pMD-28a-INP-His表面展示质粒,并将猪链球菌(SS)保护性抗原MRP、SLY基因也分别克隆至该表面展示质粒,构建pMD-INP-CdtB-OppA、pMD-INP-AfuA-OppA2、pMD-INP-MRP、pMD-INP-SLY 4种重组质粒,分别转入E.coli BL21(DE3)中诱导表达,使重组蛋白AfuA-OppA2、CdtB-OppA、MRP、SLY分别展示于E.coli BL21(DE3)的菌体表面。将灭活后的4种重组大肠杆菌按等质量比混合并添加ISA 201佐剂乳化制成表面展示二联亚单位疫苗,间隔14 d两次免疫KM小鼠,另设含rAfuA、rOppA2、rCdtB、rOppA、rMRP、rSLY的纯化蛋白亚单位疫苗免疫组、副猪嗜血杆菌-猪链球菌(HPS-SS)灭活疫苗免疫组和PBS对照组,二次免疫后的小鼠血清通过ELISA检测相应抗体和细胞因子水平。随后用HPS血清5型HN10株、血清13型ZD12株和SS血清2型M126株和血清9型GZ2株进行攻毒。结果显示:表面展示二联亚单位疫苗刺激小鼠产生的抗体和细胞因子水平与纯化蛋白亚单位疫苗组相当。用副猪嗜血杆菌5型和13型菌株分别攻毒后,表面展示二联亚单位疫苗对小鼠的保护率均为80%,其对HPS 5型菌株攻毒的小鼠保护率低于纯化蛋白亚单位疫苗(90%),但对HPS 13型菌株攻毒小鼠的保护率高于纯化蛋白亚单位疫苗(60%),且均优于HPS-SS灭活苗(80%和60%)。用SS 2型和9型菌株分别攻毒后,表面展示二联亚单位疫苗对小鼠的保护率为50%和80%,低于纯化蛋白亚单位疫苗的保护效果(100%和80%),优于HPS-SS灭活苗(50%和60%)。以上结果表明,基于表面展示技术的猪链球菌病-副猪嗜血杆菌病二联亚单位疫苗能刺激机体产生相应的抗体,其对HPS血清5型、13型和SS血清2型、9型菌株的攻击均能提供良好的交叉保护,保护效果优于HPS-SS灭活苗。本研究首次为基于表面展示技术的亚单位疫苗的研制提供实验依据,为细菌疫苗的研发提供新思路。     

Construction of Eukaryotic Expression Vector of Chicken MHCⅠα and β2m Genes and Its Expression in 293T Cells

To explore the mechanism of MHCⅠ molecule in immune response,chicken MHCⅠα and β₂m genes were cloned by PCR.Then the fragments were inserted into the eukaryotic expression vector with fluorescent protein,and the recombinant plasmids pEGFP-MHCⅠα and pmCherry-MHCⅠβ₂m were constructed.The recombinant plasmids were transfected into 293T cell with lipofectin reagent.The gene products of recombinant plasmids were mainly located to endomembrane system of the cells by fluorescence microscopy,and changed the intracellular localization of the fusion with the fluorescent protein.Moreover,the positive reactions were observed by the method of Western blotting,and the proteins had the molecular weight of 68.3 and 41.3 ku,respectively,in accord with the target proteins.The results showed that the recombinant plasmids were expressed in 293T cells with a good immunological activity,and the proteins had the binding reaction with specific antibodies.