The Construction Of Fluorescent Virus-like Particle Of Viral Structural Protein BmDNV-1VP4Fused With EGFP And Purification Of HBoV VP2

Human pathogens which lead to diseases are always evolutionary. Environmental changements and living condition variations have speeded up this process. Emergent, re-emergent viruses and novel diseases caused by those viruses are great challenges to public health as well as classic disease treatments which humans have never suffered before. For instance, the outbreak of Serve Acute Respiratory Syndrome virus (SARS) in Asia in2003, the pandemic influenza virus A (H1N1, H5N1) in2009and the newly discovered Human Boca Virus (HBoV) within the family parvoviridae in2005are typical examples. Malignant tumor which called cancer also is one of the diseases which threat human health the most. Until now, there is no efficient treatment.Looking for the valuable tools and methods to treat cancer is one of the hottest projects in scientific and medical fields. Scientists all the world are trying their best to find different ways to treat or prevent these diseases. The classical treatments of cancer, such as chemotherapy and radiotherapy, they are often accompanied with serious side effects. The newly emerged nano-biotechnology seems to have many potentials and advantages in cancer treatment. Nano-biotechnology utilizes nano materials or biological materials similar to nano particles as carrier, such as virus-like particle, to attack the abnormal cells specifically in order to cure diseases. However, the knowledge regarding to the pathway and mechanism of virus-like particle movement in cells or organisms, virus-like particle dis-assembling and re-assembling in vitro as well as the conjunction and embedding of biological effectors into the virus-like particle are not yet elucidated.This project based on the knowledge and characteristics of parvoviruses. We constructed the fusion protein expression vector of BmDNV-1Ina isolate vp4gene with the coding sequence of enhanced green fluorescent protein (EGFP) in vitro, and then produced fluorescent virus-like particle (fVLP) using Bac-to-Bac recombinant baculovirus eukaryotic expression system. These fVLPs attached with EGFP fluorescent marker can be easily used to investigate its cell penetration, movement, location, together with the dis-assembling and re-assembling properties. These results can provide useful information about anti-cancer drug designs and developments. Besides, the Human bocavirus (HBoV) vp2gene is also a good candidate for construction of VPLs, therefore, we constructed the prokaryotic expression vector and purified VP2protein to prepare polyclonal antibody for fast detection of HBoV in cells which provides us a useful tool to investigate viral proteins in cellular distribution.The main results of this project are summarized as below:1. Fluorescent virus-like particle of BmDNV-1VP4and EGFP fusion protein production1) The gene vp4of BmDNV-1and egfp gene were amplified respectively, followed by restriction enzyme digestion, ligation, transformation and screening to obtain the positive recombinants. Recombinant plasmid was transformed into E. coli DH10Bac competent cell. With the help of helper plasmid, the expression cassette was transferred and recombinant bacmid was obtained.2) Recombinant bacmid was used to transfect sf9insect cell line to obtain recombinant baculovirus. The recombinant baculovirus was then applied to infect hi5insect cell line with the optimal ratio of baculovirus stock solution to insect cell in order to acquire the EGFP-VP4fVLP. Then sucrose gradient and CsCl gradient centrifugation were used to purify the fVLP, followed with TEM observation.2. Expression and purification of HBoV VP21) HBoV structural gene vp2was amplified and inserted into prokaryotic expression vector pET-28a (+) and pMAL-c2x, the recombinant expression plasmids pET-28a (+)-VP2and pMAL-c2x-VP2were acquired and then transformed into host bacteria for protein expression and the expression levels were assayed.2) Different molecular chaperons were introduced into the host bacteria; the solubility and expression level of target protein were analyzed. The target protein was purified by affinity chromatography, which was used for polyclonal antibody production.