禽源衣原体主要外膜蛋白基因的克隆与序列分析

根据GenBank中收录的鹦鹉热衣原体主要外膜蛋白(MOMP)基因序列设计合成1对引物,以禽源鹦鹉热衣原体基因组为模板,应用PCR扩增MOMP基因,将其克隆到pUCm-T载体上,并进行序列分析。结果显示,本株禽源鹦鹉热衣原体MOMP编码基因全长为1176 bp,与国外报道的火鸡鹦鹉热衣原体TT3株的MOMP基因同源性为91.9%,与鹦鹉鹦鹉热衣原体6BC株MOMP基因的同源性为83.1%。     

鹦鹉热嗜衣原体PCR检测体系的建立

鹦鹉热是由鹦鹉热嗜衣原体(Chlamydophila psittaci,C. psittaci)引起,并可由带病鸟类传染给人类的一种人畜共患传染病.虽然有巢式PCR和多重PCR被开发用于C.psittaci的检测,但是这些方法较为繁琐.本研究应用了32株衣原体和嗜衣原体菌株,24株来自鸟类及人的肠道与呼吸道的细菌菌株,此外还使用606个禽鸟粪便、泄殖腔棉拭子及人体气管分泌物的临床样本.笔者以C.psittaci的MOMP基因(major outer membrane protein gene)为靶基因而开发了PCR检测方法.该最低能检测到100fgDNA(相当于10个衣原体)相当的鹦鹉热嗜衣原体.该靶基因区域涵盖了除C.pneumoniae、C.pecorum、C. trachomatis、C.suis和C.muridarum以外的鹦鹉热嗜衣原体所有的菌株及其他部分嗜衣原体菌株.606例临床样本中有29例鸟类粪便样本、25例泄殖腔棉拭子样本和2例人源样长检测为阳性.结果表明,该PCR检测法是检测鸟源鹦鹉热嗜衣原体的快速、敏感、有效的方法.     

猪流产衣原体CP/12株omp-1基因的克隆与表达

为观察重组MOMP蛋白的免疫活性，揭示omp-1基因(编码MOMP蛋白)在猪流产衣原体诊断和防治中的作用，用PCR法扩增猪流产衣原体omp-1基因，将特异性扩增片段克隆入pMD18-T载体，测序比较分析序列后确认为目的基因，与GenBank中4株流产衣原体omp-1基因和氮基酸序列的相似性均达99％，与其他各型衣原体代表株的相似性为71％～88％，氨基酸序列也有较大差异。将omp-1基因亚克隆到pET-32a表达载体上，再对重组表达载体转化BL21(DE3)表达宿主菌，以IPTG诱导该重组菌，结果显示该重组菌表达了融合蛋白；用猪流产衣原体多克隆抗体对表达的重组MOMP蛋白进行Western blot试验，结果显示该重组MOMP蛋白具有结合特异性抗体的活性。     

鹦鹉热衣原体real-time quantitative PCR检测方法的研究

[目的]为鹦鹉热衣原体的快速、准确检测奠定基础。[方法]根据鹦鹉热衣原体主要外膜蛋白(MOMP)基因序列设计一对特异性引物,以SYBR GreenⅠ为荧光染料,建立了鹦鹉热衣原体Real-time quantitative PCR检测方法。根据检测结果,计算样品的批内和批间变异系数(CV)。[结果]以提取的鹦鹉热衣原体DNA为模板,用引物Cps-1和Cps-2进行PCR扩增,得到长100 bp的片段。扩增产物回收纯化后,连接到pGEM-T Easy载体上并转化到大肠杆菌DH5α,菌落PCR检测筛选阳性克隆,培养后提取质粒DNA。当模板浓度范围为1.78×102~1.78×108拷贝/μl时,标准曲线相关系数达0.998;批内和批间变异系数(CV%)分别为1.30%~4.59%和5.72%~9.87%。[结论]为鹦鹉热衣原体的感染、流行调查等提供了重要的技术参考。     

Induction of a protective immune response against swine Chlamydophila abortus infection in mice following co-vaccination of omp-1 DNA with recombinant MOMP

Chlamydophila abortus is the causative agent of abortion in pigs and pregnant women. Seroconversion rates were arranged from 11% to 80% in piglets and sows in China. These very high rates illustrate the scale of the problem in China and highlight the urgent need for the development of a C. abortus vaccine. An efficacious anti-chlamydial vaccine should induce not only strong mucosal and systemic T-helper type 1 (Th1) immune response but also give a humoral response that enhances Th1 activation following infection. In order to evaluate an active immune response of a combination of the major outer membrane protein (MOMP) DNA- and protein-based vaccines, 54 BALB/c mice were randomly assigned to six groups and inoculated intramuscularly with: (i) 100 microg pcDNA::MOMP, (ii) 10 microg r-MOMP, (iii) primed with 100 microg pcDNA::MOMP and boosted with 10 microg r-MOMP, (iv) primed-boosted with a combination of pcDNA::MOMP and r-MOMP simultaneously, (v) live-attenuated 1B vaccine, (vi) 100 microg pcDNA3.1 vector. All animals were vaccinated two times at 14 days intervals. Results showed that mice given DNA and r-MOMP induced higher antibody levels, higher T cells proliferation and an elevated level of chlamydial clearance in spleen, which was equivalent to the clearance of 1B vaccine. Mice administrated the DNA-primed/MOMP-boosted approach elicited moderate antibody levels, less T-lymphocyte proliferation and lower chlamydial clearance as compared with 1B vaccine. Co-immunization with DNA- and r-MOMP vaccine may provide novel ways for active immunization strategy against swine C. abortus.     

猪流产嗜性衣原体MOMP基因的克隆及原核表达

【目的】克隆猪流产嗜性衣原体青海株主要外膜蛋白(Major outer membrane protein,MOMP)基因,并进行序列分析及原核表达。【方法】根据GenBank公布的猪流产嗜性衣原体MOMP基因的核苷酸序列,设计并合成4条特异性引物,用套式PCR方法扩增猪流产嗜性衣原体青海株MOMP基因,将其克隆入pMD18-T载体中,进行测序及序列分析。然后将MOMP基因亚克隆入原核表达载体pGEX4T-1中,在大肠杆菌BL21(DE3)中用IPTG诱导表达,对表达产物进行SDS-PAGE和Western blot检测。【结果】扩增到猪流产嗜性衣原体青海株1170bp的MOMP全长基因。序列分析结果表明,该基因与已发表的猪流产嗜性衣原体B11001株和CP/12株核苷酸同源性均为99.7%。SDS-PAGE电泳可检测到分子质量约为66ku的融合蛋白,主要以包涵体形式存在。Western-blot分析表明,重组蛋白可被猪流产嗜性衣原体抗体识别。【结论】成功克隆了猪流产嗜性衣原体青海株MOMP基因,并进行了原核表达,表达的蛋白具有抗原活性。