Recombination Analysis Of Human Bocavirus 1-4 And Construction Of Human Bocavirus 1 Related Plasmids

Objecticve1.Human bocaviruses (HBoVs) can be divided into four types, HBoVl-4. Among these four types HBoVs, only HBoVl has completed genome sequence, the other three HBoVs’ nucleotide acid sequences remains unclear. Previous studies showed that HBoV1-4 had a potential recombination relationship. So, getting the completed or nearly completed nucleotide acid sequences of HBoVl-4 has a significant meaning in analysis the genome characteristics and recombination of HBoVs.2.1nverted terminal repeat (ITR) has a significant influence on the packaging and infectivity of adeno-associated virus (AAV), at meanwhile, providing Rep and Cap proteins by exogenous plasmids can help AAV finishing packaging and release. So, constructing the HBoV1 ITR recombined plasmid and eukaryotic plasmids contain HBoV1 NS1, NP1, VP1 and VP2 have important scientific value in HBoVl related research.Subjects and Method1.Ampliation of nearly completed genome of HBoV1-4 and recombination analysis in ChinaThe single positive specimens of HBoVs were collected from children which have diarrhea symptom in Nanjing and Lanzhou. The general and anchor primers were designed by Primer premier 5.0 according to the reference strains from GenBank. Each fragment of HBoVl-4 was assembled by DNAMAN to generate the nearly completed nucleotide acid sequence. The nearly completed sequences of HBoV1-4 were done homology alignment and phylogenetic analysis. Recombination analysis of HBoV1-4 were done by RDP4.2. Construction of HBoV1 related plasmidThe pITRGFP plasmid was constructed by removing NS1, NP1, VP1 and VP2 through restriction enzyme from pIHBoVl plasmid and added EGFP which from pEGFP-N1. The pcRC plasmid was constructed by inserting the CDS region of HBoV1 into pcDNA3.1(+) vector. Then pITRGFP and pcRC plasmids were co-transfected into HEK293 cells. SDS-PAGE was used to identify the expressions of HBoV1 VP2 in order to confirm above plasmids were constructed successful.Results1. Two nearly completed sequences of HBoVl, two nearly completed sequences of HBoV2, two nearly completed sequences of HBoV3 and one nearly completed sequence of HBoV4 were amplified from specimens that were collected from Nanjing and Lanzhou. The nearly completed sequence of HBoV4 was amplified in the first in China. HBoV2-NC was the longest nearly completed sequence.2. HBoV1 and HBoV3 had low variation in every encoding regions, however, HBoV2 and HBoV4 had higher variation. We concluded that HBoV3 and HBoV4 may be the recombinant viruses from HBoVl-4. HBoV3 may be a potential recombinant of HBoVl and HBoV4, and HBoV4 may be a potential recombinant of HBoV2 and HBoV3.3. We constructed the HBoVl related plasmids pITRGFP and pcRC. HBoVl VP2 protein was expressed successfully in HEK293.Conclusions1. HBoV1-4 nearly completed nucleotide acid sequences were amplified in this study.2. HBoV1-4 have potential recombinant relationship.3. HBoV1 related plasmids pITRGFP and pcRC were constructed successfully, which had significant meaning in HBoV1 related research.