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

1. P100特异性抗重组P100抗体的制备：将重组蛋白分别与P100重链、轻链和谷胱甘肽-S-转移酶融合后，用免疫兔血清制备抗重组P100抗体，并测定其特异性活性。为了测定血清P100浓度，我们正在使用抗肽和抗重组抗体对夹心法的条件进行调整。2. P100基因快速诊断系统的开发。从正常成人外周血制备基因组DNA，经PCR扩增产物直接测定P100外显子的核苷酸序列血淋巴细胞作为模板。通过与基因组和cDNA核苷酸序列的比较，确定了五个来自核苷酸突变的同种异型氨基酸替换。虽然用与His外显子密切相关的突变特异性PCR引物对300份基因组DNA样本进行了筛查，但未发现突变。P100基因组突变的检测分析不仅需要正常人的基因组DNA，还需要反复感染患者的基因组DNA，目前我们正在制备基因组DNA样品，寻找是否存在残留的核苷酸突变。为了下一步P100基因诊断系统的建立，我们计划对PCR-SSCP基因诊断方法进行条件筛选。

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.