Cloning,Sequence Comparison And Expression Research For Adhesion Pretein 33 Of Symptomatic And Asypmtomatic Tricohmonas Vaginalis Isolates

Background and purpose: Trichomonas vaginalis (T.vaginalis) is a mono-eukaryotic protozoon, which parasitizes in people's genitourinary tract and can cause trichomoniasis. Trichomoniasis has been thought to be one of the main sexually transmitted diseases(STD). According to incompletely statistic, there are 120 million patients in the world every year. With the flowing of the world population, the number of people who are infected by T.vaginalis may be biger and biger year by year. The study showed the infection of T.vaginalis can enhance people' susceptibility to other agents of STD , so trichomoniasis is being paied more and more attention by people. T.vaginalis adhereing to the host target cells is the critical step, the contact-dependent process is mediated by adhesion protein(AP).At present, 4 adhesion proteins which are AP65, AP51, AP33 and AP23 located on membrane of T.vaginalis and participate in cell adhereing. 72 residues in the N-terminal and 24 residues in the C-terminal of AP33 have reactivity with the host cells. The mAb has the immunoreactivity with the domain within the C-terminal of the protein AP33. The research has proved the existence of cytoadherence and immunoreactivity of protein AP33 coded by ap33 gene which is single copy gene and has 3 types(ap33-1, ap33-2, ap33-3). One study revealed that the conservative sequence of ap33 gene may be used for one of genetics symbol. After a long time of evaluation, T.vaginalis may form different strain,substrain and isolate. The genetic differences among isolates of T.vaginalis were correlates with hosts records. The people infected by T.vaginalis may be trichomoniasis,carrier and so on in clinical. Cytologic and zoologic experimentes about symptomatic and asymptomatic isolates of T.vaginalis have proved that there was a obvious difference in its cytopathogenic effect determined by the difference in its genetic differences. As a useful genetic symbol, ap33 gene may give a clue about the difference in gene between symptomatic and asymptomatic isolates of T.vaginalis. Cloning,sequence comparison and expression for ap33 gene are very useful for our further study of structure and function of protein AP33. We can realize the pathogenesis of T.vaginalis and establish the basement of prevention of trichomoniasis.Our study includes two partsThe first part: Object Cloning and sequence comparison of ap33 gene for symptomatic and asymptomatic isolates. Methods We isolated T.vaginalis from symptomatic and asymptomatic patients in Sichuan of China(T.vaginalis isolations strain-2 and strain-3) and extracted total DNA from protozoa with hydroxybenzene-chloro method. According to the sequence of ap33 gene published on GenBank, 3 pairs of specific primers were designed. PCR technique was employed to amplify the ap33 gene. After the type of ap33 gene was confirmed, ap33 gene was amplified largly. After being purified, the gene were directionally cloned into plasmid pMD-18T simple vector. The transformants were screened and identified by PCR and restriction analysis; additionally, the sequence of the coding region of the ap33 gene in pMD-18T-ap33 were confirmed by DNA sequencing. The sequence of ap33 gene from T.vaginalis isolations strain-2 and strain-3 were compared with original standard from GenBank respectively. At the same time, the difference of ap33 gene between strain-2 and strain-3 was analyzed. Results The plassmid pMD-18T-ap33 for symptomatic and asymptomatic T.vaginalis isolations strain-2 and strain-3 in Sichuan of China were constructed successfully. The type of ap33 gene from 2 isolates were ap33-l equally. The size of amplified ap33 gene were 930 bp and the sequence of it had 99.9 % of homologousty with published sequence on GenBank eqally. Squence analysis between two isolates showed there were 3 base pairs of difference.The second part: Object To construct prokaryotic and eukaryotic expressional plasmid and induce to prokaryotic expression in vitro.Methods The bacteria containing plasmid pMD-18T-ap33 constructed in the first part,pUC18 and pcDNA3.1(+) were inoculated on LB/AMP plate medium respectively and cultured at 37℃for 24 hours. The single colony was transferred into LB/AMP liquid medium and cultured at 37℃over night. After extracted using Rapid Plasmid DNA Daily Mini-prep Kit, the 3 plasmids were digested fully. The target gels were cut down and the DNA fragments were reclaimed using DNA Gel Extraction Kit. The ap33 gene were subcloned into pUC18 and pcDNA3.1(+) respectively. The recombinant plasmids pUC18-ap33 and pcDNA3.1(+)-ap33 were screened and identified by PCR technique and restriction analysis. The plasmids pUC18-ap33 was induced to express by IPTG and the protein AP33 was analyzed by SDS-PAGE and Western-blot. Results The correct plasmids pUC18-ap33 and pcDNA3.1(+)-ap33 were confirmed by PCR and restriction analysis. The molecullor mass of protein induced by IPTG was about Mr 36 000 , which was as the same as the deduced one.Conclusion: In the study, symptomatic and asymptomatic isolates were obstained successfully and the ap33 gene from the two isolates were cloned and sequence analyzed. After compared with the published one, the coding sequences were compared and analyzed between the two types. Whether are there some differences between symptomatic and asymptomatic isolates in gene level? We may gain some information about Lysenko of subtype in strain. In addition, prokaryotic and eukaryotic expressional plasmid were constructed successfully and the specific protein was induce to express in vitro. These works will be used to further study about adhesion and pathogenisis of T.vaginalis and be useful for prevention of trichomoniasis.