| Objective:Human parvovirus B19 (B19) is one kind of small DNA virus which can cause a wide spectrum of human diseases. It is transmitted mainly by respiratory tract and blood, which can cause local epidemic in certain communities. B19 is reported that it is one of the important pathogens causing non-immunity hydrops, acute aplasia anemia, acute thromocytonic purpura and other diseases since we have verified the existence of B19 infection in domestic. In the same time, women and children in domestic are sensitive to B19 infection for the low level of the protective antibodies in their sera. B19 infection in those with low immunity function or immunity deficiency can be persistent and can cause chronic anemia, therefore may threaten their lives. The genome of B19 consistsof DNA of approximately 5.5kb.The genome encodes a non-structural protein, NS1, and two structural proteins that comprise the B19 capsid, the minor structural protein, VP1, and major structural protein, VP2. B19 capsids are composed mainly of VP2 protein, VP1 protein contributes less than 10% of capsid protein, and its specific region is probably exposed on virion surface. Most viral neutralizing epitopes map in the VP1 specific and VP1-VP2 junction regions. Only proteins encoded by B19 VP1 specific and VP1-VP2 junction regions can stimulate bodies to produce protective antibody. Genome of the virus varies on different regions in foreign countries. But no related investigation has been reported about the genetic variation of B19 in domestic. Gene cloning and protein expression are the important milestone in the development of molecular biology in recent years. No investigation has been reported about the genetic variation of B19 using these techniques in domestic. Using of these techniques in domestic will make the first step to change the status that diagnostic reagent depends on importation and make it possible to produce B19 viral vaccine. This study is designed to clone the VP1 unique region of B19, further to analysis the nucleotide sequence of gene group and to obtain the protein expression preliminarily. Methods: Gene segment of 480bp of VP1 unique region was amplified from aplasia anemia serum of one child diagnosed in Xijing hospital in December, 1999 by PCR using a pair of primers designed by us. Thesegment was linked with PUC19 plasmid and subsequently the VP1-PUC19 plasmid was established successfully. Sequencing, analysis, protein induction, expression and electrophoresis observation were performed. Preliminary expression is obtained. Results: (1) Compared with the published B19 VP1 unique sequences of Wi strain, two base-pair and deduced amino acid were found to be changed, contributing to 4.17% (2/480) of the total amounts of nucleotides. C was substituted by A at nt.2594, the deduced amino acid was changed from histidine to asparagine; A was substituted by G at nt.2624, the deduced amino acid was changed from asparagine to aspartic acid. Three clones were sequenced and the results had no difference. (2) Compared with the sequences of other strains published by overseas, the sequences were not necessarily the same. (3)The protein electrophoresis showed there was new protein stripe whose weight was similar to expected. Conchisions:(l)Usmg a pair of primers designed by us, 480bp gene segment of VP1 unique region was amplified by PCR in our experiment. (2) Compared with the published B19 VP1 unique sequences, two nucleotides were found to be changed. (3)The deduced amino acid were changed subsequently. (4)B19VP1 unique region gene clone and protein expression provide the good future to produce B19 diagnose reagent and protection vaccine to cure B19 infection and limit its epidemic in certain communities.
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