Site-directed mutagensis of the major antigen E2 gene of CSFV, its high level expression in Escherichia coli and the immunonicity of recombinant E2 protein / 生物工程学报

Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.

The screening and identification of an unknown virus by DNA microarray / 中华检验医学杂志

Objective In our previous study,we established DNA microarray technology for identification of medical viruses on genus levels and arboviruses on species levels.In this study,we employed these microarrays to determine the pathogen of newly isolated unknown virus in July,2006 from pig brain in Shanxi province.Methods The pathogen isolated from pig brains were inoculated in BHK21 cells.After CPE were observed,the supernatants were collected and RNA was extracted.After reverse transcription and random PCR amplification,the labeled nucleic acids were hybridized with DNA microarrays.Results The hybridization results with medical viruses DNA microarray indicated that the unknown virus belonged to Flavivirus.Combined with epidemiological investigation,we presumed that it might be a kind of arbovirus. Then the labeled specimen were further hybridized with arbovirus DNA microarray and the results confirmed that it was Japanese encephalitis virus(JBV).This coincided with PCR and sequencing analysis.Conclusions The DNA microarray we established previously could be employed to identify unknown viruses.This method provides a new method for determining new viral pathogens.

Advance in Research into the Infection of Avian Influenza Virus in Humans Crossing the Species Barrier / 微生物学通报

avian influenza virus (AIV) can not only infect avian and cause pandemics,but also result infection and initiate pandemics in humans and other mammal animals,crossing the species barrier.There have been some advance in research into the nonspecific species barrier of human respiratory tract against AIV infection and the mechanism of the infection of AIV in humans in recent years.