胸膜肺炎放线杆菌apxIC基因插入失活突变株构建及免疫原性分析

胸膜肺炎放线杆菌是引起猪传染性胸膜肺炎(APP)的呼吸道病原菌,其分泌的Apx毒素是最重要的毒力因子之一。为构建APP突变弱毒菌株,在apxIC基因下游XhoI酶切位点处插入氯霉素抗性基因(Chlr)制备转移载体,通过电转化导入APP血清10型参考菌株(D13039)进行同源重组,筛选获得apxIC基因插入突变菌株D13039C-Chlr。该突变菌株特性鉴定结果表明其溶血活性完全丧失,可正常增殖和分泌ApxI毒素,连续10次传代后基因组中插入的Chlr基因可稳定遗传,利用5个剂量(2×108CFU~2×106CFU)对每组3只小鼠腹腔攻毒结果显示突变菌株毒力较母源菌株降低至少100倍以上,将突变菌株作为弱毒活疫苗经滴鼻途径免疫仔猪后利用APP血清1型(4074)和血清10型(D13039)菌株攻毒进行免疫原性鉴定,结果显示血清1型攻毒后非免疫组4头仔猪全部死亡而免疫组4头中死亡2头,非免疫组肺损伤指数(34.4)显著高于免疫组(17.5),血清10型攻毒后非免疫组肺损伤指数(17.5)也高于免疫组(10.5),同时鼻拭子和肺组织样品的细菌重分离数及PCR检测阳性数非免疫组也明显高于免疫组,表明突变菌株作为弱毒活疫苗对仔猪具有一定的交叉免疫保护力。该突变菌株的构建为鉴定ApxI毒素活性及研制具有交叉保护活性的APP弱毒活疫苗奠定了基础。     

构建胸膜肺炎放线杆菌apxIC基因插入突变株

目的：构建胸膜肺炎放线杆菌（APP）apxIC基因插入突变菌株,以鉴定ApxⅠ毒素的生物学特性。方法：根据apxⅠ核酸序列（U05042）设计1对引物,用于自APP血清10型参考菌株（D13039）基因组DNA中扩增apxIC基因及其上下游约2．8kb的基因片段,经克隆测序后在apxIC基因下游xbI酶切位点处插入约0．9kb的氯霉素（Chl）抗性基因表达盒,构建用于转化的转移载体pUIC-Chl^r,将转移载体DNA经电转化导入APP血清10型参考菌株中进行同源重组,以获得突变菌株。结果：在含有氯霉素的培养基中经筛选获得2株丧失溶血活性的突变菌株（D13039C-Chl^r）;利用PCR和Southern blot对突变菌株鉴定,显示氯霉素抗性基因已被插入细菌基因组中。结论：利用电转化和同源重组技术构建成功APP apxIC基因插入突变菌株,为分析ApxⅠ毒素的生物学特性,进而研制APP基因工程减毒活疫苗奠定了基础。     

鸭源致病性大肠杆菌Ⅰ型菌毛pilA基因的原核表达及重组蛋白对强毒攻击的免疫保护作用

根据GenBank中人源大肠杆菌pilA基因序列,用OLIGO6.0设计PCR引物,从鸭源致病性大肠杆菌GH1.2中扩增到pilA基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将鸭源致病性大肠杆菌pilA基因正向插入原核表达载体pET-32a（+）的BamHⅠ和HindⅢ位点间,成功构建了重组表达质粒pET-32a-pilA。重组表达质粒pET-32a-pilA转化表达宿主菌BL21（DE3）,用IPTG诱导,表达出了大小约为36kD的pilA重组蛋白。表达产物用镍柱亲和层析纯化,与等量弗氏佐剂混合制备pilA重组蛋白疫苗,分别在1日龄、8日龄时两次对雏鸭进行免疫,二免后2周测定鸭血清中的ELISA抗体效价,并以10⁹PFU同源菌株GH1.2攻毒,根据攻毒后鸭的死亡率、E.coli分离率和各组织器官的病变等级来判定pilA重组蛋白的免疫保护效果。结果pilA重组蛋白免疫鸭的血清中ELISA抗体效价为1∶12800,全菌灭活苗免疫组的血清ELISA抗体效价为1∶200;同源菌株攻毒后,pilA重组蛋白免疫保护组鸭的死亡率、E.coli分离率和各组织器官的病变程度均比攻毒对照组下降且差异显著或极显著,与全菌灭活苗免组比较差异不显著。表明pilA重组蛋白对同源菌株GH1.2的感染具有一定的保护效果     

鸭源致病性大肠杆菌Ⅰ型菌毛pilA基因的原核表达及重组蛋白对强毒攻击的免疫保护作用

根据GenBank中人源大肠杆菌pilA基因序列,用OLIGO6.0设计PCR引物,从鸭源致病性大肠杆菌GH1.2中扩增到pilA基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将鸭源致病性大肠杆菌pilA基因正向插入原核表达载体pET-32a（+）的BamHⅠ和HindⅢ位点间,成功构建了重组表达质粒pET-32a-pilA。重组表达质粒pET-32a-pilA转化表达宿主菌BL21（DE3）,用IPTG诱导,表达出了大小约为36kD的pilA重组蛋白。表达产物用镍柱亲和层析纯化,与等量弗氏佐剂混合制备pilA重组蛋白疫苗,分别在1日龄、8日龄时两次对雏鸭进行免疫,二免后2周测定鸭血清中的ELISA抗体效价,并以10⁹PFU同源菌株GH1.2攻毒,根据攻毒后鸭的死亡率、E.coli分离率和各组织器官的病变等级来判定pilA重组蛋白的免疫保护效果。结果pilA重组蛋白免疫鸭的血清中ELISA抗体效价为1∶12800,全菌灭活苗免疫组的血清ELISA抗体效价为1∶200;同源菌株攻毒后,pilA重组蛋白免疫保护组鸭的死亡率、E.coli分离率和各组织器官的病变程度均比攻毒对照组下降且差异显著或极显著,与全菌灭活苗免组比较差异不显著。表明pilA重组蛋白对同源菌株GH1.2的感染具有一定的保护效果     

肠炎沙门氏菌基因工程减毒株的免疫效果及安全性评价

[背景] 相较于灭活疫苗和弱毒疫苗,沙门氏菌（Salmonella）基因工程减毒活疫苗具有的优越性逐渐显现,研究也不断深入。[目的] 探究肠炎沙门氏菌G9菌株的4株基因缺失株G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）的免疫效果及生物安全性。[方法] 以肠炎沙门氏菌基因缺失菌株G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）、G9（ΔssrABhilAhilD）及其亲本菌株G9的最佳免疫剂量接种小鼠后,利用间接ELISA法、流式细胞术、MTT法、小鼠攻毒试验及倾注平板法等对各缺失株的免疫效果和安全性进行评价。[结果] G9（ΔhilD）诱导血清IgG抗体效价最高,G9（ΔhilD）和G9（ΔssrABhilAhilD）产生肠黏膜IgA抗体效价最高,4株缺失菌诱导IL-4、IL-10、IFN-γ、TNF-β、MCP-1细胞因子的能力与亲本株G9差异不显著（P>0.05）,产生的CD4⁺/CD3⁺、CD8⁺/CD3⁺ T细胞比率呈上升趋势,而且G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）最高;G9（ΔssrABhilAhilD）诱发的脾淋巴细胞增殖指数最高;免疫小鼠后4株缺失株对G9攻毒提供的保护率为80%-100%,而且小鼠肝脏、脾脏及小肠绒毛无明显病理变化,对鼠伤寒沙门氏菌攻毒提供的保护率为50%-80%;接种后12 d,小鼠肝脏、脾脏及小肠中定殖的菌株能基本清除,而且在体外能连续稳定传代30代。[结论] G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）对小鼠的免疫效果和生物安全性均良好,有成为肠炎沙门氏菌基因工程减毒活疫苗的可能。     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σ^E因子控制的cry1Aa基因表达。     

猪链球菌4型疫苗用菌株的筛选

【背景】猪链球菌4型（Streptococcus suis serotype 4,SS4）分离率日益升高,对养殖业和公共卫生安全造成严重危害,目前尚无有效的SS4疫苗。【目的】筛选致病力强、抗原性好、遗传性状稳定的SS4疫苗菌种。【方法】以7株SS4分离株（代号为A1—A7）为受试菌株,通过累积法测定菌株半数致死量（LD₅₀）,ELISA测定免疫小鼠血清中IgG效价,攻毒保护试验测定免疫保护率,并采集小鼠脏器观察病理组织学变化。再连续传代培养受试菌株,分别对第10、20、30代菌株进行致病性和抗原性试验。【结果】A1—A7菌株对小鼠的LD₅₀分别为2.19×10⁸、1.76×10⁸、1.83×10⁸、1.01×10⁸、4.05×10⁸、1.19×10⁸和9.03×10⁷ CFU。二免7 d后,A1、A2、A3、A4、A6、A7免疫组IgG效价分别为1：1 600、1：1 600、1：3 200、1：6 400、1：3 200和1：6 400,免疫保护率分别为30%、30%、50%、70%、60%和80%,而且A4、A7免疫组小鼠组织病变较其余4组轻微。体外传至30代后,A4菌株的LD₅₀上升至3.81×10⁸ CFU,IgG效价下降至1：1 600,免疫保护率下降至40%,而A7菌株的LD₅₀上升至2.49×10⁸ CFU,IgG效价和免疫保护率稳定保持为1：6 400和80%,而且A7免疫组小鼠的组织病变较A4免疫组轻微。【结论】A7菌株（原始编号为HBgu18-4）具有强致病力和良好的抗原性,而且遗传性状均一、稳定,可作为SS4制苗候选菌株。     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

猪胸膜肺炎放线杆菌血清1型RTX毒素I的N-端表达多肽具有良好的免疫原性

ApxI外毒素是猪胸膜肺炎放线杆菌(APP)最重要的毒力因子,为了研究其N端多肽的免疫原性,分别将apxIA基因的全长编码区(apxIA,3146bp)及其5′端1140bp的片段(apxIA5)克隆到原核表达载体pET28a,经IPTG诱导后在大肠杆菌中实现了表达,表达产物ApxIA和ApxIAN均以包涵体的形式存在,Westernblot检测证实两种表达产物均具有免疫反应性。将纯化的重组蛋白(rApxIA和rApxIAN)和提取的天然毒素ApxI(nApxI)分别经腹腔免疫BALBc小鼠,于免疫前、免疫2周和4周后分别检测了ELISA抗体和毒素中和抗体水平,结果表明,rApxIAN免疫组的ELISA抗体显著低于rApxIA免疫组和nApxI免疫组,但rApxIAN免疫组血清中和试验中测定的溶血素单位与rApxIA及天然nApxI免疫组没有显著差异。第二次免疫2周后,用1个LD50的APP血清1型J101株和2型标准菌株攻击试验动物,rApxIAN免疫组对血清1型和2型菌株的保护率分别为80%和100%。     

鼠伤寒沙门菌小RNA RybB及伴侣蛋白Hfq对孔蛋白OmpD的表达调控

【目的】探究小RNA (small RNA, sRNA) RybB和伴侣蛋白Hfq对沙门氏菌孔蛋白OmpD表达的调控作用。【方法】以鼠伤寒沙门菌(Salmonella Typhimurium, STM)为研究对象,将含有编码β-半乳糖苷酶的lacZ报告基因的pCE40质粒转入ompD基因单缺失菌株中以获得lacZ报告菌株;在此基础上,利用P22噬菌体转导技术分在lacZ报告菌株中构建rybB全序列缺失、hfq全序列缺失、hfq点序列敲除和hfq序列截短以获得双突变实验菌株,以及rybB全序列缺失和hfq全序列缺失的三突变实验菌株。通过β-半乳糖苷酶活性试验和RT-qPCR探究sRNA RybB及伴侣蛋白Hfq对孔蛋白OmpD表达的调控作用。【结果】成功在lacZ报告菌中构建rybB全序列缺失、hfq全序列缺失、hfq点序列敲除和hfq序列截短等双缺失实验菌株,以及rybB全序列缺失和hfq全序列缺失的三突变实验菌株。与野生型(wild type, WT)菌株相比,在lacZ报告菌株中,hfq基因截短为87个氨基酸序列的突变株中的OmpD蛋白活性下调了2.16%,其余实验株OmpD蛋白的β-半乳糖苷酶活性均呈上升趋势。与WT菌株相比,实验菌株ompD基因转录水平除了STM LT2∆ompD::lacZΔhfq65的上调不具有显著性外,其余实验菌株均显著(P<0.05)上调,其中lacZ报告菌株、rybB全序列缺失和hfq全序列缺失的三突变实验菌株ompD基因转录水平上调最明显,为1.83倍。【结论】ompD基因的转录及蛋白表达主要受hfq基因和sRNA RybB的负反馈调节;Hfq的远端面在对ompD基因的转录抑制中起关键作用。通过多个基因突变菌株的构建,阐述了sRNA伴侣蛋白Hfq与孔蛋白OmpD的相互作用关系,探索了Hfq对ompD的调控关键区域,丰富了sRNA的调控理论。     

The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae: evidence from mutational analysis

The involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogen Actinobacillus pleuropneumoniae in virulence was investigated using haemolysin-deficient mutants constructed by a mini-Tn10 mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini-Tn10 were cloned and sequenced. In the non-haemolytic mutant, the transposon had inserted in apxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In the apxI operon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved in A. pleuropneumoniae pathogenicity. The non-haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.     

malT knockout mutation invokes a stringent type gene-expression profile in Actinobacillus pleuropneumoniae in bronchoalveolar fluid

Background  

Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min.     

Isolation and Characterization of LPS Mutants of Actinobacillus pleuropneumoniae Serotype 1

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae. Received: 23 December 1996 / Accepted: 19 February 1997     

双组分系统rcsC基因影响禽致病性大肠杆菌的致病性及相关生物学特性

【目的】双组分系统Rcs感受外界环境变化,并调控细菌的适应性及生存等。本文探讨Rcs双组分系统传感器激酶RcsC对禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)相关生物学特性及致病性的影响。【方法】采用Red同源重组的方法构建rcsC基因缺失株,并利用互补质粒构建互补株,然后比较野生株、基因缺失株与互补株的生长特性、运动性、生物被膜、凝集沉淀能力、致病力及毒力基因转录水平的差异。【结果】rcsC基因缺失不影响APEC的生长速度,然而,缺失RcsC导致APEC的运动能力升高、生物被膜形成能力降低和凝集能力增强。凝集试验结果显示rcsC基因有助于APEC的凝集沉降。细胞黏附入侵结果表明,rcsC在APEC侵袭DF-1细胞过程中发挥作用,而对黏附能力无影响。动物感染试验结果表明rcsC基因缺失能显著降低APEC的毒力。荧光定量PCR检测结果表明,rcsC基因缺失株中ompA、aatA、fyuA和luxS基因的转录水平均显著降低,而fimC和tsh基因的转录水平显著升高。【结论】RcsC参与调控APEC的运动性、生物被膜形成、凝集沉降和致病力。     

Protection of Mice Against Challenge with Homologous and Heterologous Serovars of Actinobacillus pleuropneumoniae After Live Vaccination

Protective immune responses and the virulence of Actinobacillus pleuropneumoniae (APP) have been attributed, in part, to toxins (Apx) produced by the bacterium. A mutant of the serovar 7 strain HS93 (HS93Tox⁻), lacking the genes encoding the structural toxin ApxA and the post-translational activating protein ApxC, but retaining the genes required for secretion ApxB and ApxD, was isolated and shown to be attenuated in a mouse model. A plasmid vector system was developed and used to express the ApxA gene from within the HS93Tox⁻ strain. The resulting strain, HS93Tox⁻/pIG-T1K, expresses the Apx structural protein in a non-activated form. HS93Tox⁻/pIG-T1K was shown to be attenuated in a mouse model and to be capable of inducing Apx-specific antibodies, which were boosted on re-inoculation. Live vaccination of mice with HS93Tox⁻/pIG-T1K offered protection against homologous wild-type serovar 7 challenge, and also heterologous challenge with a serovar 1 strain. This is in contrast to vaccination with the HS93Tox⁻ strain, which failed to protect mice against a heterologous challenge. Received: 13 April 1998 / Accepted: 16 June 1998     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.     

Isolation of a DNA polymerase I (polA) mutant of Rhizobium leguminosarum that has significantly reduced levels of an IncQ-group plasmid

A population of Tn5 mutagenised Rhizobium leguminosarum cells was screened for mutants affected in protein secretion by introducing a plasmid carrying the Erwinia chrysanthemi prtB gene and screening for mutants defective in secretion of the protease PrtB. One such mutant (A301) also appeared to be defective in secretion of the R. leguminosarum nodulation protein NodO. Genetic analysis showed that the defect in A301 was caused by the Tn5 insertion. However the DNA sequence adjacent to the site of Tn5 insertion had significant homology to the Escherichia coli polA gene, which encodes DNA polymerase I. The mutant A301 showed increased sensitivity to ultraviolet light, a characteristic of polA mutants of E. coli. The apparent defect in secretion by A301 was due to a large decrease in the copy number of the IncQ group replicon on which prtB and nodO were cloned and this decreased the total amounts of PrtB or NodO protein synthesised and secreted by the polA mutant. The polA mutant had a lower growth rate than the parent strain on both rich and minimal media, but there was no obvious effect of the polA mutation on the symbiosis of R. leguminosarum bv. viciae with pea.     

Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10(5) higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence-associated regulator also in S. serovar Choleraesuis.