几株b型流感嗜血杆菌多糖结合物的免疫原性检定

Research on PCR determining of type b HaemophUus influenzae strains and immunogenicity of polysaccharide conjugates

目的从分子水平检定b型流感嗜血杆菌，研究不同菌株荚膜多糖结合物的免疫原性。方法提取基因组，通过型特异和荚膜型基因特异引物，利用PCR检定b型流感嗜血杆菌；不同纯化多糖分别与破伤风类毒素（TT）进行耦联结合，结合物原液经稀释免疫小鼠，通过两针免疫，采血进行免疫效力测定。结果5株b型流感嗜血杆菌通过PCR法均能获得预期大小的型特异（482bp）和荚膜型（343bp）基因片段，BLAST分析显示，各菌之间型特异和荚膜型序列比对，其同源性均为100％，各菌型特异和荚膜型序列分别与GenBank X78559．1和M19995．1序列比对，同源性分别为99％和100％；ELISA检测显示，4株不同菌株来源的荚膜多糖结合物（PRP-TT）的小鼠免疫效力差异无统计学意义。结论通过PCR法从分子水平检定b型流感嗜血杆菌，纯化的不同荚膜多糖结合物小鼠免疫原性基本一致。可供不同Hib多糖结合物免疫原性的研究参考数据。

Objective To determine Haemophilus influenzae type b strains in molecular level using PCR, and to study the immunogenicity of capsular polysaccharide conjugates in mice. Methods Extracting genomes using bacterial DNA extract kit from Haemophilus influenzae type b strains, and PCR for determining the strains through serotyping-specific and capsular genotyping primers respectively. Various capsular polysaccharides conjugated TT respectively, and the conjugates were administered subcutaneously to mice through dilution. After vaccination with two doses, blood samples were collected for the detection of antibody levels to polyribosylribitol phosphate （PRP）, the capsular polysaccharide of Hib. Results All five Haemophilus influenzae type b strains contain type-specific（482 bp） and capsular type （343 bp）DNA fragment through PCR detecting. The DNA fragments were sequenced. BLAST show that these sequences are 100% homology comparing the above strains respectively, and are 99% and 100% homology comparing the Gen- Bank X78559.1 and M19995.1 respectively. The immunogenicity of mice from various capsular polysaccharide conjugates （PRP-TT） was not significantly different by ELISA detecting. Conclusion Through PCR, Haemophilus influenzae type b strain can be determined in molecular level. The immunogenicity of mice from purified capsular polysaccharide conjugates was not different. The study provides a detection means for the features and heredity stability of Haemophilus influenzae type b strain and reference data for the immunogenicity of different polysaccharide conjugates in vaccine research and development and production.