致肾盂肾炎大肠杆菌粘附基因群的克隆及其抗血清的制备

以柯斯质粒pHC79为载体构建致肾盂肾炎大肠杆菌代表株E.coli J96染色体基因文库,获得两个能够表达粘附特性的重组柯斯质粒.进一步用鸟枪法亚克隆到载体pACYC184,得到三个阳性重组体.其中pCT10/E.coli K-12 p678-54能够稳定表达P菌毛和MRHA,分子量为14.6kb.用该克隆子免疫新西兰白兔,抗血清用带有载体质粒的受体菌(pACYC184/E.coli K-12 P678-54)吸收后,经菌毛粗提物的SDS-聚丙烯酰胺凝胶电泳及Western blotting和血凝抑制试验检测,其特异性仅针对致肾盂肾炎大肠杆菌的粘附基因群,可用于临床菌株的鉴定.

MOLECULAR CLONING OF THE ADHERENCE GENE CLUSTER FROM UROPATHOGENIC ESCHERICHIA COLI AND PREPARATION OF ITS ANTISERA

A genomic library of the uropathogenic E. coli J96 was constructed by using cosmid pHC79 as cloning vector. Two positive recombination cosmids which could express the adherence characters were acquired. From both cosmids a EcoRI fragment was subcloned into the vector pACYC184 by shot-gun method. Three colonies were found which exhibited MRHA and production of P Pili. One of them , pCT10/E. coli K-12 P678-54, was about 14. 6 kb and was used to prepare the antisera. After absorption with pACYC184/E. coli K-12 P678-54 for three times, the antisera were revealed specific against the adherence gene cluster of uropathogenic E. coli by the SDS-PAGE and Western blotting of the P pilus crude extracts and the hemagglutination inhibition test.