多杀性巴氏杆菌兔体内差异表达基因及其突变株构建的研究
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摘要
多杀性巴氏杆菌（Pasteurella multocida，P. multocida）能够引起多种动物感染，导致急性、败血性传染病，包括禽霍乱、牛出血性败血症、猪肺炎和猪萎缩性鼻炎、兔出血性败血症。多杀性巴氏杆菌属于革兰氏阴性菌，巴氏杆菌科巴氏杆菌属，主要以荚膜抗原和菌体抗原区分血清型，荚膜抗原有5群，分别为A、B、D、E、F群；菌体抗原分为16个型，分别为1、2、3、4、5、、16。本菌呈世界性分布，已给养殖业造成巨大的经济损失，是重点防控的疫病之一。
本研究以兔源多杀性巴氏杆菌C51-17菌株为研究对象，采用选择性捕获转录序列技术对C51-17菌株分别在感染兔肝脏组织和BHI培养基培养的条件下的差异表达基因进行筛选。结果表明：通过选择性捕获转录序列技术鉴定到31个基因，按照其编码蛋白的功能分为5大类：新陈代谢和应激蛋白，细胞表面蛋白，调节蛋白、转运蛋白和3个未知功能蛋白。利用Real-timeRT-PCR对差异表达基因进行转录水平的验证，结果表明：purF、lon、dnaB、ftsQ和glpT等5个的基因与BHI培养基中生长的C51-17菌株的基因转录水平相比，C51-17菌株在感染兔肝脏组织中5个基因的转录水平均上调，上调了1.61～13.55倍。
将C51-17菌株热休克蛋白70基因（dnaK）克隆到原核表达载体pPro-EXHTb进行序列分析和原核表达，诱导表达重组DnaK蛋白，进行抗原性和免疫原性分析，测定其生物学活性。结果表明：C51-17菌株的dnaK基因与其他多杀性巴氏杆菌菌株dnaK基因在核苷酸水平的同源性均在99.2%以上，与其他种属的细菌的dnaK基因的同源性在82.3%～97.5%；成功表达了C51-17菌株重组DnaK蛋白，分子量为70kDa，与预期大小一致，以可溶性的形式存在；Western blot试验证明，重组DnaK蛋白具有良好的抗原性；纯化的DnaK蛋白分别按照50μg/只和150μg/只免疫ICR小鼠，对致死性剂量的C51-17菌株的保护性分别为1/5和2/5；重组DnaK蛋白具有粘附细胞活性，并能抑制多杀性巴氏杆菌对细胞的粘附作用；将dnaK基因克隆到真核表达载体pEGFP-C1构建真核表达质粒转染细胞，检测蛋白在细胞中的定位，结果表明：DnaK蛋白主要定位于细胞的细胞质中。
采用正向筛选同源重组技术构建C51-17菌株ΔdnaK突变株和ΔaroA突变株，利用PCR对突变株进行鉴定，分析其遗传稳定性、生长特性和致病性。结果表明：成功构建了多杀性巴氏杆菌ΔdnaK突变株和ΔaroA突变株，连续传代20代，它们遗传稳定；ΔdnaK突变株和ΔaroA突变株与亲本菌的体外生长曲线表明，ΔdnaK突变株和ΔaroA突变株与亲本菌C51-17的体外生长曲线基本一致。ΔdnaK突变株和ΔaroA突变株对ICR小鼠的致病实验结果表明：腹腔注射ICR小鼠，ΔdnaK突变株在剂量为1.0×10~2CFU时对小鼠有致死性，而ΔaroA突变株在剂量为1.0×10~6CFU时对小鼠无致死性；而亲本菌C51-17在剂量为1.0×10~2CFU对小鼠是完全致死性的。ΔdnaK突变株对小鼠的致病性无明显减弱，ΔaroA突变株对小鼠的致病性是减弱的。
本研究采用选择性捕获转录序列技术筛选C51-17菌株在感染兔肝脏中和BHI培养的条件下的差异表达基因，利用Real-time RT-PCR对差异表达基因进行转录水平的验证。同时，采用正向筛选同源重组技术构建C51-17ΔdnaK突变株和ΔaroA突变株。多杀性巴氏杆菌正向筛选同源重组构建突变株的技术与选择捕获转录序列技术鉴定到的多杀性巴氏杆菌兔体内差异表达基因相结合，为进一步研究多杀性巴氏杆菌致病机理和减毒新型基因工程疫苗奠定基础。
Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowlcholera in poultry, hemorrhagic septicemia in cattle, atropic rhinitis in swine and snuffles in rabbits.Strains of P. multocida are normally designated on the basis of the capsular serogroup and somaticserotype. There are five serogroups (A, B, D, E, and F) based on capsule specificity, and16somaticserotypes (1–16) based on lipopolysaccharide antigens. The bacterial is a worldwide distribution and animportant disease to prevent and control, causing economical losses to the animal breeding industry.
The differentially expressed gene profile of P. multocida C51-17strain in infected rabbit livers wasidentified and compared with that from in vitro culture by selective capture of transcribed sequences. Atotal of31genes were identified, of which28encoded enzymes for amino acid biosynthesis andmetabolism, intermediary metabolism, and energy metabolism, or proteins for regulatory adaptiveresponses, general microbial stress response, transport proteins and secreted proteinases. Three wereunknown, novel genes. Five genes representing different categories were chosen randomly and verifiedby real-time reverse transcriptase-polymerase chain reaction analysis. All Genes (purF, lon, dnaB, ftsQ,and glpT) were upregulated by P. multocida in infected rabbit livers, with changes ranging from1.61-fold to13.55-fold when compared with in vitro cultures.
The dnaK gene of heat shock protein70(DnaK) of P. multocida strain C51-17was cloned intoprokaryotic expression vector pPro-EXHTb, transformed into BL21(DE3) and induced by IPTG. Theantigenicity and immunogenicity and biologic activity of recombinant DnaK protein were analysised.The results showed that dnaK gene at the nucleotide level had homology with other P. multocida strainsavboe99.2%, and82.3%to97.5%with other species of bacteria. rDnaK was purified by the Ni-NTAaffinity chromatography. The results of SDS-PAGE and western blot showed that DnaK was amolecular weight of70kDa in soluble form and recognized by positive serum of P. multocida with goodantigenicity. The immunogenicity of rDnaK was determined in ICR mice and showed that the micewere immuned with50μg and150μg rDnaK protein and survived1/5and2/5at the lethal dosage of50CFU of P. multocida, respectively. The adhesion and inhibition activity of rDnaK was detected andindicated that protein was adhered on Vero cells and inhibition of adherence of P. multocida to Verocells. The dnaK gene cloned into eukaryotic expression plasmid pEGFP-C1, transformed into Vero cellsand indicated that DnaK was localizated in cytoplasm.
Suicide recombinant plasmid of pBC-DnaK-Km and pBC-aroA-Km were constructed with kanamycingene and transformed into P. multocida by electroporation. The ΔdnaK and ΔaroA mutant strains of P.multocida were screened by kanamycin resistance and chloromycetin sensitivity and identified by PCR.Its virulence of ΔdnaK and ΔaroA mutant strain was tested in mice by intraperitoneal injection. TheΔdnaK and ΔaroA mutant strains of P. multocida C51-17were successfully constructed. Growth rateofΔdnaK and ΔaroA mutant strains were similar to the wild-type strain in vitro. The virulence testindicated that ΔdnaK mutant strain was no significant attenuated and the death was found in miceinoculated at a dosage of1.0×10~2CFU, and ΔaroA mutant strain was significant attenuated and no death was found in mice even inoculated at a dosage of1.0×10~6CFU. The virulence of ΔaroA mutant strain ofP. multocida was highly attenuated in mice.
The differentially expressed genes of P. multocida C51-17strain were identified by selective captureof transcribed sequences and verified by real-time reverse transcriptase-polymerase chain reactionanalysis. The ΔdnaK and ΔaroA mutant strains of P. multocida C51-17were constructed using positivescreening homologous recombination technology. This study will provide a molecular basis for furtherstudy of the pathogenesis and attenuated vaccine of P. multocida.
本研究以兔源多杀性巴氏杆菌C51-17菌株为研究对象，采用选择性捕获转录序列技术对C51-17菌株分别在感染兔肝脏组织和BHI培养基培养的条件下的差异表达基因进行筛选。结果表明：通过选择性捕获转录序列技术鉴定到31个基因，按照其编码蛋白的功能分为5大类：新陈代谢和应激蛋白，细胞表面蛋白，调节蛋白、转运蛋白和3个未知功能蛋白。利用Real-timeRT-PCR对差异表达基因进行转录水平的验证，结果表明：purF、lon、dnaB、ftsQ和glpT等5个的基因与BHI培养基中生长的C51-17菌株的基因转录水平相比，C51-17菌株在感染兔肝脏组织中5个基因的转录水平均上调，上调了1.61～13.55倍。
将C51-17菌株热休克蛋白70基因（dnaK）克隆到原核表达载体pPro-EXHTb进行序列分析和原核表达，诱导表达重组DnaK蛋白，进行抗原性和免疫原性分析，测定其生物学活性。结果表明：C51-17菌株的dnaK基因与其他多杀性巴氏杆菌菌株dnaK基因在核苷酸水平的同源性均在99.2%以上，与其他种属的细菌的dnaK基因的同源性在82.3%～97.5%；成功表达了C51-17菌株重组DnaK蛋白，分子量为70kDa，与预期大小一致，以可溶性的形式存在；Western blot试验证明，重组DnaK蛋白具有良好的抗原性；纯化的DnaK蛋白分别按照50μg/只和150μg/只免疫ICR小鼠，对致死性剂量的C51-17菌株的保护性分别为1/5和2/5；重组DnaK蛋白具有粘附细胞活性，并能抑制多杀性巴氏杆菌对细胞的粘附作用；将dnaK基因克隆到真核表达载体pEGFP-C1构建真核表达质粒转染细胞，检测蛋白在细胞中的定位，结果表明：DnaK蛋白主要定位于细胞的细胞质中。
采用正向筛选同源重组技术构建C51-17菌株ΔdnaK突变株和ΔaroA突变株，利用PCR对突变株进行鉴定，分析其遗传稳定性、生长特性和致病性。结果表明：成功构建了多杀性巴氏杆菌ΔdnaK突变株和ΔaroA突变株，连续传代20代，它们遗传稳定；ΔdnaK突变株和ΔaroA突变株与亲本菌的体外生长曲线表明，ΔdnaK突变株和ΔaroA突变株与亲本菌C51-17的体外生长曲线基本一致。ΔdnaK突变株和ΔaroA突变株对ICR小鼠的致病实验结果表明：腹腔注射ICR小鼠，ΔdnaK突变株在剂量为1.0×10~2CFU时对小鼠有致死性，而ΔaroA突变株在剂量为1.0×10~6CFU时对小鼠无致死性；而亲本菌C51-17在剂量为1.0×10~2CFU对小鼠是完全致死性的。ΔdnaK突变株对小鼠的致病性无明显减弱，ΔaroA突变株对小鼠的致病性是减弱的。
本研究采用选择性捕获转录序列技术筛选C51-17菌株在感染兔肝脏中和BHI培养的条件下的差异表达基因，利用Real-time RT-PCR对差异表达基因进行转录水平的验证。同时，采用正向筛选同源重组技术构建C51-17ΔdnaK突变株和ΔaroA突变株。多杀性巴氏杆菌正向筛选同源重组构建突变株的技术与选择捕获转录序列技术鉴定到的多杀性巴氏杆菌兔体内差异表达基因相结合，为进一步研究多杀性巴氏杆菌致病机理和减毒新型基因工程疫苗奠定基础。
Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowlcholera in poultry, hemorrhagic septicemia in cattle, atropic rhinitis in swine and snuffles in rabbits.Strains of P. multocida are normally designated on the basis of the capsular serogroup and somaticserotype. There are five serogroups (A, B, D, E, and F) based on capsule specificity, and16somaticserotypes (1–16) based on lipopolysaccharide antigens. The bacterial is a worldwide distribution and animportant disease to prevent and control, causing economical losses to the animal breeding industry.
The differentially expressed gene profile of P. multocida C51-17strain in infected rabbit livers wasidentified and compared with that from in vitro culture by selective capture of transcribed sequences. Atotal of31genes were identified, of which28encoded enzymes for amino acid biosynthesis andmetabolism, intermediary metabolism, and energy metabolism, or proteins for regulatory adaptiveresponses, general microbial stress response, transport proteins and secreted proteinases. Three wereunknown, novel genes. Five genes representing different categories were chosen randomly and verifiedby real-time reverse transcriptase-polymerase chain reaction analysis. All Genes (purF, lon, dnaB, ftsQ,and glpT) were upregulated by P. multocida in infected rabbit livers, with changes ranging from1.61-fold to13.55-fold when compared with in vitro cultures.
The dnaK gene of heat shock protein70(DnaK) of P. multocida strain C51-17was cloned intoprokaryotic expression vector pPro-EXHTb, transformed into BL21(DE3) and induced by IPTG. Theantigenicity and immunogenicity and biologic activity of recombinant DnaK protein were analysised.The results showed that dnaK gene at the nucleotide level had homology with other P. multocida strainsavboe99.2%, and82.3%to97.5%with other species of bacteria. rDnaK was purified by the Ni-NTAaffinity chromatography. The results of SDS-PAGE and western blot showed that DnaK was amolecular weight of70kDa in soluble form and recognized by positive serum of P. multocida with goodantigenicity. The immunogenicity of rDnaK was determined in ICR mice and showed that the micewere immuned with50μg and150μg rDnaK protein and survived1/5and2/5at the lethal dosage of50CFU of P. multocida, respectively. The adhesion and inhibition activity of rDnaK was detected andindicated that protein was adhered on Vero cells and inhibition of adherence of P. multocida to Verocells. The dnaK gene cloned into eukaryotic expression plasmid pEGFP-C1, transformed into Vero cellsand indicated that DnaK was localizated in cytoplasm.
Suicide recombinant plasmid of pBC-DnaK-Km and pBC-aroA-Km were constructed with kanamycingene and transformed into P. multocida by electroporation. The ΔdnaK and ΔaroA mutant strains of P.multocida were screened by kanamycin resistance and chloromycetin sensitivity and identified by PCR.Its virulence of ΔdnaK and ΔaroA mutant strain was tested in mice by intraperitoneal injection. TheΔdnaK and ΔaroA mutant strains of P. multocida C51-17were successfully constructed. Growth rateofΔdnaK and ΔaroA mutant strains were similar to the wild-type strain in vitro. The virulence testindicated that ΔdnaK mutant strain was no significant attenuated and the death was found in miceinoculated at a dosage of1.0×10~2CFU, and ΔaroA mutant strain was significant attenuated and no death was found in mice even inoculated at a dosage of1.0×10~6CFU. The virulence of ΔaroA mutant strain ofP. multocida was highly attenuated in mice.
The differentially expressed genes of P. multocida C51-17strain were identified by selective captureof transcribed sequences and verified by real-time reverse transcriptase-polymerase chain reactionanalysis. The ΔdnaK and ΔaroA mutant strains of P. multocida C51-17were constructed using positivescreening homologous recombination technology. This study will provide a molecular basis for furtherstudy of the pathogenesis and attenuated vaccine of P. multocida.
引文
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