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

1.血清P100水平快速诊断系统的研制。P100特异性抗重组P100抗体的制备：将重组蛋白与P100重链或轻链和谷胱甘肽- s -转移酶融合后复制，从免疫兔血清中制备抗重组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。P100基因快速诊断系统的研制。P100基因的异型分析：P100外显子的核苷酸序列是直接从PCR扩增的产物中确定的，基因组DNA是从正常成人外周血淋巴细胞作为模板制备的。通过与基因组和cDNA核苷酸序列的比较，鉴定了由核苷酸突变产生的5个异型氨基酸取代。虽然使用与His外显子密切相关的突变特异性PCR引物筛选了300个基因组DNA样本，但尚未发现突变。对P100基因组突变的调查分析，不仅需要正常感染患者的基因组DNA，也需要复发感染患者的基因组DNA。目前，我们正在准备基因组DNA样本，寻找剩余核苷酸突变的存在。对于P100基因诊断系统的下一步，我们计划筛选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.