Development of a double-antibody sandwich ELISA targeting the receptor binding domain of TcdB toxin of ST11 type Clostridium difficile of porcine origin / 生物工程学报

Clostridium difficile is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type C. difficile is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting C. difficile of porcine origin, leaving hidden dangers for the prevention and control of C. difficile. The aim of this study was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type C. difficile of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (κ). Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution. The negative cutoff OD450 was 0.152, and no cross-reaction with 13 strains of non-ST11 type C. difficile was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type C. difficile in pig industry.

Serological and molecular epidemiology of Japanese encephalitis virus infections in swine herds in China, 2006–2012

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing viral encephalitis in humans and reproductive disorder in swine. JEV is prevalent throughout China in human; however, spatiotemporal analysis of JEV in Chinese swine herds has not been reported previously. Herein, we present serological and molecular epidemiological results and estimates of prevalence of JEV infections among swine herds in various regions of China. The results suggest that JEV infections are widespread and genotype I and III strains co-exist in the same regions. Therefore, there is an urgent need to monitor JEV infection status among swine herds in China.

Analysis on the non-structural protein genes of avian influenza viruses subtype H5N1 isolated from chicken in China / 中国人兽共患病学报

Fourteen H9N2 avian influenza viruses (AIV) were isolated from sick chickens in China from 1998 to 2008. The sequences of the Non-structural(NS) gene of these isolates were determined by RT-PCR and sequencing, and the entire ORF sequences of NS1 and NS2 protein were obtained.-The homology of these nucleotide sequences and the putative amino acid sequences were compared with several classic reference viruses of H9N2. These isolates were proved to be highly homologous in NS gene (92.9%-99.9% identity) and all belonged to A/Chicken/Beijing/1/1994-like group in the Asia bird-swine branch of allele A of HS gene phylogenetic tree.-According to this study and previous reports of other researchers, NS gene of H9N2 subtype AIV in chickens of China is genetically stable and there is no enough evidence to support the establishment of other sub-lineages in chickens.

Cloning and expression of the σC gene of avian reovirus / 中国兽医学报

The σC gene of ARV S1133 was designed to amplify by reverse transcription chain reaction(RTPCR).The σC gene was inserted into the vector pMD19-T,identified by PCR method and restriction enzyme,and sequenced.It showed that the insert cloned gene fragment was the σC gene of ARV.Then the gene was inserted intc the pET32a(+) and indicated that fusion expression vector pET32a-σC was constructed.The recombinant fusion protein was highly expressed in E.coli BL21 induced by 1.0 mmol/L IPTG for 5 hours in the form of inclusion bodies.The weight of recombinant fusion protein molecular is 54 000.Western-blot with ARV antibodies against the fusion protein showed the recombinant protein has a favourable reactivity.