LaVerda D, Byrne G I
Department of Medical Microbiology and Immunology, University of Wisconsin, Madison 53706, USA.
J Clin Microbiol. 1997 May;35(5):1209-15. doi: 10.1128/jcm.35.5.1209-1215.1997.
As a requisite for a physiological and immunological investigation, reagents were developed that facilitated the identification and purification of Chlamydia trachomatis hsp10 (chsp10). Monoclonal antibodies that specifically recognize chsp10 were generated with multiple-antigen peptides (MAPs) to promote recognition of Chlamydia-specific epitopes. MAP2, containing amino acids 54 to 69 of the hsp10 sequence, elicited strong antibody responses after immunization of BALB/c mice. Monoclonal antibodies from several cloned hybridomas reacted on immunoblots with an approximately 15-kDa chlamydial protein and recombinant chsp10. Because of its strict specificity for chsp10, monoclonal antibody M1.2 was selected for routine use. M1.2 reacted by immunoblot with the hsp10s of several C. trachomatis strains but not with Chlamydia psittaci hsp10 or Escherichia coli homolog GroES, suggesting that M1.2 recognizes a species-specific epitope. Recombinant chsp10 was purified by immunoaffinity chromatography with M1.2. For large-scale purification, chsp10 was appended with a C-terminal six-histidine tag for purification by nickel chelate affinity chromatography. The hypA gene encoding the chsp10 of C. trachomatis serovar E/Bour was cloned into the pQE-60 vector (QIAGEN, Inc.) following PCR amplification from genomic DNA. E. coli DH5 transformants were screened for chsp10 expression by colony immunoblotting with M1.2, were tested for nickel matrix binding, and were sequenced. The sequence of serovar E/Bour chsp10 was found to be closely homologous to those of hsp10s of other chlamydiae. Purified chsp10 and specific anti-chsp10 monoclonal antibodies will be useful for investigating the biological and immunological roles of hsp10 in chlamydial infections.
作为生理和免疫学研究的一项必要条件，研发了有助于沙眼衣原体热休克蛋白10（chsp10）鉴定和纯化的试剂。利用多抗原肽（MAPs）产生了特异性识别chsp10的单克隆抗体，以促进对衣原体特异性表位的识别。包含hsp10序列第54至69位氨基酸的MAP2在免疫BALB/c小鼠后引发了强烈的抗体反应。来自几个克隆杂交瘤的单克隆抗体在免疫印迹上与一种约15 kDa的衣原体蛋白和重组chsp10发生反应。由于其对chsp10具有严格的特异性，选择单克隆抗体M1.2用于常规使用。M1.2通过免疫印迹与几种沙眼衣原体菌株的hsp10发生反应，但不与鹦鹉热衣原体hsp10或大肠杆菌同源物GroES发生反应，这表明M1.2识别一种种特异性表位。用M1.2通过免疫亲和层析纯化重组chsp10。为了大规模纯化，在chsp10的C末端附加一个六组氨酸标签，以便通过镍螯合亲和层析进行纯化。从基因组DNA进行PCR扩增后，将编码沙眼衣原体血清型E/布尔hsp10的hypA基因克隆到pQE - 60载体（QIAGEN公司）中。通过用M1.2进行菌落免疫印迹筛选大肠杆菌DH5转化体的chsp10表达，检测其与镍基质的结合情况并进行测序。发现血清型E/布尔chsp10的序列与其他衣原体hsp10的序列密切同源。纯化的chsp10和特异性抗chsp10单克隆抗体将有助于研究hsp10在衣原体感染中的生物学和免疫学作用。
