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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 sp32 / OY-TES-1 mRNA is expressed in normal tissues and the expression of sp32 / OY-TES-1 mRNA. Methods The total RNA was extracted from 40 normal tissues (19 samples), cDNA was synthesized by reverse transcription - polymerase (RT - PCR) and the quality of cDNA was detected by ordinary PCR. The target gene sp32 / OY - FES - 1 and housekeeping gene HPRT plasmid were constructed and prepared standard The expression of sp32 / OY-TES-1 and HPRT was detected by real-time quantitative PCR (TaqMan) and optimized reaction system. The expression of sp32 / OY-TES-1 mRNA was expressed as OY-TES-1 / HPRT . The expression of sp32 / OY-TES-1 protein in various normal tissues was detected by the prepared OY-TES-1 polyclonal antibody combined with immunohistochemistry. Results The positive rate of sp32 / OY-TES-1 mRNA in normal tissues was 72.50% (29/40) .2.The relative expression of sp32 / OY-TES-1 mRNA in normal tissues ) Was 0.0001 ~ 0.4950, showing a lognormal distribution.3. The relative expression of sp32 / OY-TES-1 mRNA in testes was 44.9, which was 91-1000 times that of other normal tissues.4. Immunohistochemistry ) Were detected in a variety of normal tissues and cells tested (10 testicular tissue, 8 colon, 7 liver, 6 skeletal muscle, 5 gastric, 5 lens, 4 sperm, 3 peripheral blood leukocytes and 1 kidney). No positive expression of OY-TES-1 protein was found in the other 4 sperms and 1 renal tubule. Conclusion The expression of sp32 / OY-TES-1 mRNA is widely expressed in various normal tissues. The expression of sp32 / OY-TES-1 mRNA varies with different tissues. Testicular tissue is the most abundant tissue expressed in normal tissues. The sp32 / OY-TES-1 protein is more restricted than its mRNA expression.