人细小病毒B19 VP1独特区基因变异的研究

目的：获取人细小病毒B19(HPV B19)VP1独特区基因,并进行序列测定及变异分析,为研制诊断试剂及预防疫苗创造条件。方法：应用聚合酶链反应（PCR）技术从1例急性特发性血小板减少性紫癜患儿的血清中扩增HPV B 19VP1独特区基因片段,将其克隆至pGEM-T easy载体,转化大肠杆菌DH5α,筛选阳性克隆,测定目的基因的序列。结果：成功地扩增到HPV B19 VP1独特区全长基因,长度为705个核苷酸,测定结果与Genbank中Gallinella G和Venturoli S所发表的HPV B19 VP1独特区全长基因序列比较,有2处核苷酸发生突变,但所编码的氨基酸均未发生变化。结论：（1）HPV B19 VP1独特区有基因变异;（2）构建了HPV B19 VP1独特区基因的重组质粒,所获实验结果为深入研究奠定了基础。

Genetic diversity of human Parvovirus B19 VP1 unique region

OBJECTIVE: Human Parvovirus B19 (HPV B19) is a small (23 nm), non-enveloped DNA virus found in 1974. It has been proved that HPV B19 is associated with a variety of childhood diseases, such as erythema infectious, transient aplastic crisis, aplastic anemia, idiopathic thrombocytopenic purpura and arthropathy, etc. There have been no any effective vaccines to prevent HPV B19 infection so far. The HPV B19 genome is composed of 5.6 kb single strand DNA. This genome encodes a nonstructural protein NS1, two structural proteins VP1 and VP2. Most neutralizing linear epitopes of HPV B19 cluster in the VP1 unique and VP1-VP2 junction regions. Only proteins encoded by genes of the VP1 unique and VP1-VP2 junction regions can stimulate bodies to produce protective antibodies. Aim of the present study was to get the VP1 unique region gene of HPV B19 and to analyze the genetic diversity so as to further study its function and application. METHODS: The VP1 unique region gene of HPV B19 was amplified from the serum of a child with idiopathic thrombocytopenic purpura by PCR. The purified PCR product was cloned into pGEM-T easy vector and transfected into the host strain E. coli (DH5 alpha). Positive clones were chosen and then the target gene was sequenced. RESULTS: The target gene sequence of HPV B19 VP1 unique region was amplified and cloned successfully. It had 705 nucleotides. Compared with the relevant sequences published in Genbank, the sequencing results were revealed with two nucleotides changes in the HPV B19 VP1 unique region, but their coding amino acid were not changed. CONCLUSION: It is suggested that genetic diversity exists in the VP1 unique region of HPV B19. Construction of the recombinant plasmid of HPV B19 VP1 unique region gene might benefit to further study.