摘要
目的了解恙虫病东方体Karp株相对分子质量为47蛋白的表达,检测其抗原性和免疫原性。方法采用PCR扩增恙虫病东方体Karp株相对分子质量为47蛋白成熟肽全基因序列并TA克隆,酶切PCR产物构建pGEX-47原核表达质粒,转化BL21,IPTG诱导表达相对分子质量为47重组蛋白,并检测其抗原性及免疫原性。结果1.扩增PCR产物约1300bp,测序结果与相对分子质量为47蛋白基因序列相同;2.构建原核表达质粒pGEX-47,纯化的重组蛋白SDS-PAGE分析在相对分子质量为47处,薄层扫描分析表达率为15.8%;3.Dot blot分析显示其有抗原性,小鼠免疫实验证实其有免疫原性。结论扩增相对分子质量为47蛋白的成熟肽全基因序列,在原核细胞得到表达,表达产物具有抗原性和免疫原性。
Objective To know the expression of 47kD protein of orientia tsutsugamushi（Ot） Karp strain and test its immunogenicity and antigenicity .Methods Forty-seven kD protein gene that code the whole may peptide was amplified from the genomic DNA of Ot.Karp strain by PCR and constructed to a recombinant plasmid pGEX-47.The E.coli cells BL21 were transfomed with pGEX-47,then the transformants were induced to express the recombinant 47kD protein by IPTG.The antigenicity and immunogenicity were identified by Dot Blot metbod and the experiment of immunizing mice respectively.Results 1.The PCR product of 47kD protein gene of Ot.Karp strain was obtained and its sequence was the same as the published 47kD protein gene.2.The E.coli expression plasmid pGEX-47 constructed was identified containing the 47kD protein gene fragment by restrication analysis and PCR identifcation.and an expression band about 47kD was detected by SDS-PAGE of the purified product from GST-fusson protein expressed by pGEX-47.3.Dot blot analysis and the experiment of immunizing mice testified its antigenicity and immunogenicity.Conclusions The 47kD protein gene of Ot Karp Strain is amplified that code the whole mat peptide,which can be expressed in prokaryotic cells and the expressed protein has antigenicity and immunogenicity.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2005年第9期853-854,i0001,共3页
Journal of Applied Clinical Pediatrics
基金
广州市重点攻关课题资助(2000-J-001-01)
