Development of a rapid diagnosis system for the recurrent infectious constitutions.

1.血清P100水平快速诊断系统的建立。P100特异性抗重组P100抗体的制备：将P100重链或轻链与谷胱甘肽-S转移酶融合的重组蛋白复制后，用免疫兔血清制备抗重组P100抗体，并检测其活性。2.建立P100基因快速诊断系统。P100基因同种异型分析：以正常成人外周血淋巴细胞基因组DNA为模板，直接从PCR扩增产物中确定P100外显子的核苷酸序列。通过与基因组和c DNA核苷酸序列的比较，确定了5个来自核苷酸突变的同型氨基酸替换。虽然使用与His外显子密切相关的突变特异性聚合酶链式反应对300个基因组DNA样本进行了筛选，但这些突变仍未被发现。对于P100基因突变的分析，不仅需要正常患者的基因组DNA，也需要反复感染患者的基因组DNA。目前，我们正在准备基因组DNA样本，寻找是否存在残留的核苷酸突变。对于下一步的P100基因诊断系统，我们正在计划筛选适合于PCR-SSCP基因诊断方法的条件。

1.Development of a rapid diagnosis system for estimation of serum P100 levels.Preparation of the P100 specific anti recombinant P100 antibodies : After reproduction of the recombinant proteins that fused with heavy or light chains of P100 and glutathion-S-transferase, the anti-recombinant P100 antibodies were prepared form immunized rabbit serum and assayd those specific activities. To determine of the serum P100 concentrations, we are performing the adjustment for the conditions of the sandwich method using anti-peptid and anti-recombinant antibodies.2.Development of a rapid diagnosis system for the P100 gene.Allotype analysis of the P100 gene : The nucleotide sequences of the P100 exons were direct determine from PCR amplified products that genome DNA was prepared from a normal adult peripheral blood lymphocyte as a template. Five allotypic amino acid substitutions derived from nucleotide mutations were identified comparisons with genomic and cDNA nucleotide sequences. Although screen for 300 genome DNA samples using mutation specific PCR primers that closely related to His exon, the mutations has not been find out. To the analysis for the survey of P100 genomic mutation, it is needed not only normal but recurrent infectious patients genome DNA.At the present, we are preparing those of genome DNA samples and searching for the presence of remained nucleotide mutatios. To the next step for the gene diagnosis system of P100, we are planning to screening conditions for the PCR-SSCP gene diagnostic method.