Gene Cloning, Prokaryotic Expression And Monoclonal Antibody Preparation Of Human Epididymal Sperm Protein P34H

Mammalian spermatozoa are highly differentiated by the time they leave the testis. Nonetheless, they do not yet have the ability to move progressively or to interact with an oocyte and they gain these abilities during the epididymal transit along the caput, corpus and cauda segment. Many studies have reported that epididymal secretory products are involved in the sperm maturation and epididymal proteins have been shown to interact with spermatozoa and to modify their surface properties. Some of sperm surface proteins change their location from one membrane domain to another during sperm maturation and other sperm surface proteins are altered, masked or replaced by new proteins of epididymal origin. During epididymal maturation sperm membrane lipids undergo distinct physical and chemical alterations. Collectively, these modifications, mainly including the acquisition of sperm motility and fertilizing ability, are referred to as epididymal sperm maturation.P34H, a member of the short-chain dehydrofenase/reductase(SDR) superfamily, is acquired on the acrosomal cap of human spermatozoon during its maturation arising within epididymis. Antibodies against this human protein inhibit sperm-zona pellucida binding in vitro, without affecting motility, the ability to acrosome react or sperm-egg plasma membrane fusion. P34H appears to be involved in one of the absolute prerequisites of fertilization, i.e. sperm-zona pellucida binding. And P34H is proposed to be used as a marker of epididymal sperm maturation in humans. Research results also revealed that the occurrence of low concentration of sperm protein P34H were significant amongst the idiopathic infertile male population. Therefore the level of sperm protein P34H is proposed to be an auxiliary diagnostic marker for male infertility. Furthermore, recent report that human kidney dicarbonyl/L-xylulose reductase (DCXR) and P34H are probably the same protein, indicates that P34H is also associated with nephrosis. Therefore there is a need to acquire large amounts of purified P34H and its antibody, not only for the establishment of an immunological method to detect P34H, but also for exploring its function in male reproductivesystem and application in the diagnosis of male infertility.In the first trial, human epididymal sperm protein P34H gene coding region was amplified from human corpus epididymis tissue by RT-PCR and cloned into pGEM-T vector. There was a difference of two bases between the sequence of the amplified DNA fragment and that of human P34H cDNA previously reported. After homologous comparison in GenBank with the help of BLAST software it was shown that the coding region of P34H was identical to that of human kidney dicarbonyl/L-xylulose reductase (DCXR). So it was confirmed that DCXR and P34H are the same protein in terms of their cDNA coding sequence. Meanwhile the nucleotides sequence amplified from human corpus epididymis was submitted to GenBank and had been acceptd (Accession No. AF 515623).To determine the distribution of the P34H gene expression, in the second trial access RT-PCR was performed to amplify P34H gene from total RNA of the different segment epididymis and testis tissues with fi-actin as an inner control. According to the ratio of P34HI $ -actin, it was concluded that P34H was preferentially expressed in the human corpus epididymis but much lest in the caput, cauda epididymis and testis tissue. This was in agreement with the conclusion drawn by Northern blot analysis in the published paper.In the third part of this study, digestion of pGEM-T/P34H with BamH I and Hind III released the 0.75kb P34H fragment which was subcloned into the same restriction sites of the pQE-30 expression vector. The recombinant expressive vector designated pQE-30/P34H was transformed into competent E.coli DH5oc for the identification of correct construction. Then the plasmid pQE-30/P34H was transformed into the competent M15(pREP4) cells to induce the expression of the recombinant protein P34H on the reduction of IPTG. The inductive conditions were a...