Expression of a novel pyridoxal kinase mRNA splice variant,PKH-T, in human testis

Aim: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa. Methods: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template. The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results: A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1 069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different develop-mental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients. Conclusion: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility. (Asian J Androl 2004 Jun; 6: 83-91)

人睾丸特异性新基因hT279单克隆抗体制备及其应用

目的构建人睾丸特异性新基因hT279(GenBank登录号BC016750)的原核表达载体,纯化融合蛋白并以其为抗原免疫Balb/c小鼠制备抗人睾丸特异性hT279蛋白单克隆抗体(mAb)并进行特性鉴定。方法用PCR方法得到睾丸特异性新基因hT279并克隆至pET32a原核表达载体中,转化大肠杆菌BL21诱导融合蛋白表达;获得的可溶性蛋白经亲和层析纯化、SDS-PAGE鉴定后,免疫Balb/c小鼠,应用淋巴细胞杂交瘤技术制备抗hT279 mAb,并通过间接ELISA、Western blot和免疫组织化学法对mAb进行特性鉴定。结果实现了hT279的原核表达,获得了1株稳定分泌抗hT279 mAb的杂交瘤细胞株4B2,抗体亚型为IgG2b(κ),效价达到1×104。Western blot鉴定表明,该mAb在人正常睾丸蛋白中相对分子质量约为22 000处检测到特异条带。免疫组织化学显示hT279蛋白主要在正常人睾丸的精母细胞及圆形精子细胞中表达,而在男性不育患者睾丸组织中表达消失。结论成功制备出1株抗hT279的mAb 4B2,为进一步研究hT279在人类精子发生中的功能和男性不育症的诊断提供了特异的检测工具。