上海地区户尘螨的分离培养及Der p 2基因的克隆与表达

克隆化培养上海地区户尘螨,获得不同编码序列的重组Der p 2变应原进行当地人群血清特异性IgG分析,探讨其应用价值。从过敏性疾病患者床褥采集分离户尘螨,采用封闭式培养系统进行培养,分离培养单个孕螨获得克隆化的螨群。用RT-PCR的方法扩增不同螨克隆的Der p 2编码基因,插入原核表达载体pET-19b在大肠杆菌内进行表达,所获得的蛋白经Dot blot和ELISA检测其抗原性及与人群血清IgG的反应水平。结果显示,成功对户尘螨种群进行克隆化培养。获得14个核苷酸多样性位点,对应的两个不同序列的重组Der p 2蛋白与鼠单抗1D8的亲和力不同,但与上海地区人群IgG结合力相似(r=0.864)。检测不同地区尘螨主要变应原的序列多样性,有助于开发更有针对性的疫苗组分,以及特异性保护性IgG的筛选。

Isolation of Dermatophagoides pteronyssinus in Shanghai and cloning and expression of the Der p 2 gene

This study is to colonize the Dermatophagoides pteronyssinus in Shanghai,and to acquire the nucleotide polymorphism of Der p 2 in this area.According to the specific IgG level of people living in Shanghai to the rDer p 2,the value of application of rDer p 2 with specific sequences was estimated.D.pteronyssinus were collected from the beds of patients with allergic rhinitis and cultured in a system closed of from the environment.Pregnant mites were singly picked up and cultured in small wells separately,so that some subclones could be obtained 6 weeks later.Der p 2 fragments were acquired with RT-PCR,inserted into pET-19b and the proteins were expressed in E.coli strain BL21(DE3)plysS.Dot blot and ELISA were used to check the allergenicity of the recombinant allergens and their reactivity to serum IgG of the population.We successfully colonized the D.pteronyssinus in Shanghai area.14 nucleotides polymorphism sites were got and two corresponding recombinant Der p 2 were with different affinity to murine mAb 1D8,but with similar reactivity to serum IgG of Shanghai population.To examine the sequence polymorphism of major allergens of dust mites is beneficial to the development of more pertinent vaccines and the screening for protective IgG.