Gene Engineering Expressing And Antibody Preparation Of Human Testis Sperm Protein SP22

In 1997, Klinefelter GR reported nitroimidazole and its derivative had effect on rats'fertility. Further researches showed that these chemical materials could strip a protein from sperm's surface and result in asthenospermia. Klinefelter GR named this protein sperm protein 22(SP22). SP22 is generally expressed in all tissues. The expression of SP22 in different specieses was high homology. SP22 has 90% homology between oncogene DJl and transcription factor RS. And rat's contraception associated protein 1, CAP1 is identical to SP22. SP22' s general expression and high homology between species demonatrate SP22 may be encoded by housekeeping gene, be very important for vital movement. So the gene sequence of SP22 has been keeping invariable in long-term evolution.SP22 has variable function. The combination of sperm and ovum on ovum's zona pellucida can be interrupted by SP22 antibody in vitro or vivo. SP22 is supposed to participate acrosomal reaction. The research of SP22's structure reveals that SP22 is an enzyme, which has enzymolysis on ovum's zona pellcida and assists sperm to penetrate into ovum. The high homology and protein structure like DJ1 also illutrate SP22 is like an enzyme. DJ1 resided in brains participates anti-oxydation mechanism, which can combine with oxidation metabolic product and degradate them. Mutation of DJ1 made oxidation products accumulated in brain, and also resulted in diseases. And DJ1 is only an enzyme that has a direct correlation with Parkinson's disease. Study showed just one mutation of amino acids in DJ1 could result in P arkinson's disease in early age. SP22 is similar to transcription factor RS, which existed intranuclear and outside. During mitotic division, some SP22 protein enter cell nuclear. Not like other intranuclear proteins, SP22 has no intranuclear localization sequence. SP22 coheres to other intranuclear proteins in order to enter the nuclear. In nuclear, RS combines with neucleoprotein RBS rather than nucleic acid directly. SP22 is supposed as a regulatory factor, rather than transcription factor, participating cellproliferation. When SP22 is binding with ras factor, SP22 obtains great carcinogenic activities, which can induce rats NIH3T3 cell canceration.SP22's multifunction evokes people's reconstruction. Further researches are going on to investigate SP22's function and property. But all experiments were restricted on animal. Human's testis SP22 and its localization in male reproductive system is not be studied home and abroad. SP22 participates acrosomal reaction. Its quantity on the surface of sperm was directly influenced by some reproductive intoxicant, which could result in male infertility. So in our experiment, we selected human testis tissue, clone its SP22 gene by RT-PCR, transferred it into pGEM-T vector and DNA was sequenced. Its sequence was compared with animal homologous genes. Homology is as high as reported. It's human's SP22 in human testis that has been cloned and its sequence similar to other species's. Similarly, we found its conservation thoughout evolution. The amplificated human testis SP22 was subcloned into express vector pET-28(a), pHIL-Sl, transferred into gene engineering bacterium BL21, Pichia Pastoris GS115 to express the human testis gene SP22. The expressed protein was detected in recombinant E coli's cytorrhyctes and recombinant GS115's culture supernatant. We compared the prokaryotic expression system with the eukaryotic expression system. The expressed recombinant protein SP22 was purified. Prokaryotic and eukaryotic expressed recombinant protein have a six histidine in the protein's N end called His(+)-tag. They could be purified by nickel ion affinity chromatograph. These purified recombinant proteins was identified and contrasted. We used the prokaryotic expressed protein SP22 as antigen to immune rabbit for preparation of rabbit anti-human SP22 polyclonal antibody. The antibody titer had been analysed. The antibody could specificly react with expressed SP22 and native SP22. And then the antibody was used to detect SP22's localization in human testis and sperm.On the whole, these experiment shows that human testis SP22 gene sequence has great high homology with other species SP22; that SP22 is vital for life activities. The two gene engineering expression germs have been successfully constructed and compare the advantages and disadvantages between two expression systems. Rabbitanti-human sperm protein SP22 polyclonal antibody was prepared, which also can react to native SP22 in human reproductive system. The antibody was used to detect localization of native SP22 in human reproductive system.