Gene Clone And Sequence Analysis Of Human Parvovirus B19 NS Region Of Chinese Strain

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. Many a investigation finds B19 is associated with transient aplastic crisis, erythema infectiosum, acute thromocytonic purpura, non-immunity fetus hydrops and other diseases. Infectious rate of B19 is high in domestic population. B19 infection in those with low immunity function or immunity deficiency can be persistent and can cause chronic anemia, and in some serious cases may threaten their lives. The genome of B19 consists ofDNA of approximately 5.6kb. The genome encodes a non-structural protein (NS), and two structural proteins(VP1 and VP2). Genome of the virus varies on different regions in foreign countries. B19 NS protein has cytotoxicity and can induce apoptosis. Variations of NS are related with pathogenicity of B19. But few related investigations have been reported about the genetic variation of B19 NS in domestic. In recent years molecular cloning technique progresses rapidly and there are many methods in molecular cloning. Molecular cloning technique is extensively used with the using of polymerase chain reaction technique. PCR products can be directly cloned into pGEM-T vector. This study is designed to clone the NS gene of Chinese B19 strains, further to analysis the nucleotide sequence of the NS gene and discuss the variation of Chinese B19 strains.Methods: Four pairs of inner primers and a pair of full-length primers are designed by us. The inner primers can be used to amplify four crossing parts of B19 NS gene (NSA 569bp nt436-992, NSB 582bp nt982-1151, NSC 603bp nt!537-2127, NSD 561bp nt2092-2640) and the full-length primers can be used to amplify B19 NS in full-length(2096bp nt406-2501).B19 NS gene was amplified from aplasia anemia sera of three children diagnosed in Xijing hospital in 2001 by PCR using the inner primers. NS genes of three Chinese B19 strains were sequenced and analysised. NS gene was linked with pGEM-T plasmid andsubsequently the NS-pGEM-T plasmid was established successfully. NSD-pGEM-T was established using the same method.Results: (l)Compared with the published B19 NS sequence of Au strain, four base-pairs and deduced four amino acids were found to be changed in the three Chinese B19 strains XA17, XA18, XA20. T was substituted by C at nt!082, the deduced amino acid was changed from phenylalanine to serine in XA17; T was substituted by C at nt!619, the deduced amino acid was changed from valine to alanine in XA18; T was substituted by C at nt521, the deduced amino acid was changed from leucine to serine in XA20; G was substituted by C at nt!552, the deduced amino acid was changed from leucine to serine in XA20. We found no substitution in the region nt2092-2451. (2)New methods using PCR to detect B19 DNA were established and proved to be special and susceptible. (3)The agarose electrophoresis showed there were stripes whose base-pairs were expected. NS-pGEM-T and NSD-pGEM-T were established successfully.Conclusions: (1)Using four pairs of crossing inner primers and a pair of full-length primers designed by us, expected gene NS segments were amplified by PCR in our experiment. Special and susceptible new methods using PCR to detect B19 DNA were established. (2)Compared with the published B19 Au strain NS sequence, four nucleotides were found to be changed in XA17, XA18, XA20 strains. The deducedamino acids were changed subsequently. (3) Chinese B19 stranins NS gene were sequenced and cloned and thus provided the good future to produce B19 DNA PCR diagnose reagent, prevent and cure B19 infection and limit its epidemic in certain communities.