精子蛋白sp32/OY-TES-1 mRNA及其蛋白在正常组织表达的探讨

THE STUDY OF SPERM PROTEIN sp32/OY-TES-1 mRNA AND PROTEIN EXPRESSION IN HUMAN NORMAL TISSUES

目的了解正常组织中sp32/OY-TES-1 mRNA的表达量是否存在差异以及该蛋白的表达情况,为sp32/OY-TES-1表达谱增加更多信息。方法提取40例(19种)正常组织总RNA,逆转录-聚合酶合成cDNA,普通PCR检测cDNA质量;构建目的基因sp32/OY-FES-1及看家基因HPRT质粒,制备标准品,绘制标准曲线,建立实时定量PCR(TaqMan)方法和优化反应体系,检测组织中sp32/OY-TES-1和HPRT的表达,sp32/OY-TES-1 mRNA的表达量以OY-TES-1/HPRT表示。通过制备的OY-TES-1多克隆抗体,结合免疫组织化学检测sp32/OY-TES-1蛋白在多种正常组织中的表达。结果正常组织中sp32/OY-TES-1 mRNA阳性率为72.50%(29/40);2.非睾丸正常组织中sp32/OY-TES-1 mRNA的相对表达量值(目的基因/看家基因)为0.0001～0.4950,呈对数正态分布;3.睾丸组织中sp32/OY-TES-1 mRNA的相对表达量为44.9,是其他正常组织的91～1000倍;4.免疫组织化学(IHC)显示在所检测的多种正常组织和细胞中(10例睾丸组织、8例结肠、7例肝、6例骨骼肌、5例胃、5例晶状体、4例精子、3例外周血白细胞和1例肾),除4例精子和1例肾组织中的肾小管外,其余均未见OY-TES-1蛋白阳性反应。结论sp32/OY-TES-1 mRNA广泛地表达在各种正常组织,表达量因组织不同而异;睾丸组织是所检测的正常组织中其mRNA表达量最高的组织。而sp32/OY-TES-1蛋白比其mRNA更具有限制性表达的特点。

Objective To investigate whether the expression of sp32/OY-TES-1 mRNA is various in human normal tissues, study the expression of its protein, and provide more information about sp32/OY-TES-1 expressive profile. Methods Extraction of total RNA from 40 cases of human normal tissues included 19 different types, cDNA was synthesized by reverse transcriptase and tested by conventional polymerase chain reaction （PCR）. With the construction of sp32/OY-TES-1 and HPRT plasmid for preparation of standard products, standard curves and the protocol of quantitative real-time PC R （TaqMan method） were generated, and reaction system was optimized. The expressions of sp32/OY-TES-1 and HPRT were tested respectively; OY- TES-1/HPRT was used to evaluate the expression level of sp32/OY-TES-1 mRNA. Sp32/OY-TES-1 protein that expressed in normal tissues was tested by immunohistochemistry staining with pelyclonal antibody against OY-TES-1 protein. Results 1 .The expressive frequency of sp32/OY-TES-1 in a panel of normal tissues was 72.50 % （29/40） ; 2. The value of OY-TES-1/HPRT was used for the quantification of sp32/OY-TES-1 mRNA expression, which was between 0.000 1 and 0.459 0 in human non-testis normal tissues and showed in the range of logarithmic normal distribution. 3. The relative quantitative expression of sp32/OY-TES- 1 mRNA in human testis tissue was 44.9, which was 91-1 000 times higher than that in other normal tissues tested. No positive target protein reaction was found in testis, colon, liver, skeletal muscle, stomach, lens, or leukocyte except in the sperm and the renal tubule of kidney. Conclusion With an extensive expression in human normal tissues, the expression level of sp32/OY- TES-1 mRNA was diverse. Of a panel of normal tissues tested, testis tissue was found with the highest expression, sp32/OY-TES-1 protein had a stricter expression in normal tissue than its mRNA.