Rescue of the recombinant infectious bronchitis virus with the ectodomain region of H120 spike glycoprotein / 病毒学报

To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus (IBV) Beaudette strain, the ectodomain region of the Spike gene (1,302 bp) of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA. This recombinant was designated as BeauR-H120(S1). BeauR-H120(S1) was directly used as the DNA template for the transcription of viral genomic RNA in vitro. Then, the transcription product was transfected into Vero cells by electroporation. At 48 h post-transfection, the transfected Vero cells were harvested, and passaging continued. A syncytium was not observed until the recombinant virus had passed through four passages. The presence of rBeau-H120(S1) was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR. Western blot analysis of rBeau-H120 (S1)-infected Vero cell lysates demonstrated that the nucleocapsid (N) protein was expressed, which implied that rBeau-H120(S1) could propagate in Vero cells. The TCIDs0 and EIDs0 data demonstrated that the titer levels of rBeau-H120(S1) reached 10(590+/-0.22)TCID50/mL and 10(6.13+/-0.23)EID50/mL in Vero cells and 9-day-old SPF chicken embryos, respectively. Protection studies showed that the percentage of antibody-positive chickens, which were vaccinated with rBeau-H120(S1) at 7 days after hatching, rose to 90% at 21 days post-inoculation. Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41 strain (103EID50/chicken). This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis.

Secreted expression of nonstructural protein gene 3ABC of foot-and-mouth disease virus in Sf9 cells and activity analysis / 生物工程学报

Entire 3ABC sequence of FMDV containing a 6 x his tag coding sequence at the N-terminal was obtained through PCR amplification using a pair of specific primers, subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence and finally constructed recombinant plasmid of pMel-3ABC. After co-transfected the recombinant plasmid and linearized Bac-N-Blue DNA into Sf9 insect cell under intermediary agent of the Cellfectin, the result showed that we have already acquired recombinant baculovirus by screen of plaque assay and identification of PCR. Though the recombinant baculovirus infecting the Sf9 cells again, experiments indicated that 3ABC gene could express in insect cells and the expressed protein was secreted in the supernatant of Sf9 cell culture possessing favourable biological activities detected by adopting two methods of SDS-PAGE and Western blot. The result verified that the protein could respond with sera derived from FMDV infected animals, but have no responsibility with sera derived from health animals and vaccinated animals detected by indirect ELISA using antigen of expressed protein after purification with Ni-NTA his bind resin. Therefore, this study has established a solid foundation for establishing an effective diagnosis method to discriminating the FMDV infected animals from vaccinated animals.

Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system / 生物工程学报

To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system.

Prediction of Secondary Structure and B Cell Epitope for Capsid Protein of SVDV / 中国生物工程杂志

The secondary structure of Capsid protein was predicted by the methods of Chou-Fasman,Garnier-Robson and Karplus-Schultz based on the sepuence of capsid protein gene of Swine Vesicular Disease Virus (SVDV) and hydrophilicity. Surface probility plot and antigenic index for capsid protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-wolf, respectively, Combining the results according to these methods, the B cell epitopes for capsid protein of SVDV were predicted. The results showed that there are much flexible region such as coil region and turn region in capsid protein of SVDV, there are more predominant B cell epitopes in VP1 than in VP2 and VP3. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of SVDV.

Expression of recombinant plasmid pcDNA3.1/P12X3C with multi-genes of foot-and-mouth disease virus in BHK-21 cells / 生物工程学报

In order to obtain the gene P12X3C of Foot-and-Mouth Disease Virus (FMDV) that includes full length P1, 2A, 3C and a part of 2B, the site mutation strategy was used. After being digested by Kpn I and Xba I respectively, the gene P12X3C was cloned into the pcDNA3.1 (+) expression vector. The recombinant plasmid was checked by restriction enzyme analysis and nucleic acid sequencing, and then named pcDNA3.1/P12X3C. Further, BHK-21 cells was transfected with pcDNA3.1/P12X3C by using lipoid. The proteins of Foot-and-Mouth Disease Virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy. The result shows the gene P12X3C is cloned into eukaryotic expression plasmid, and the recombinant eukaryotic expression plasmid pcDNA3.1/P12X3C could express proteins of Foot-and-Mouth Disease Virus in BHK-21 cells, which have immunocompetence. This study demonstrates that delivery of a recombinant eukaryotic expression plasmid containing P12X3C coding regions results in the assembly of FMDV capsid structures, which will offer experimental base to DNA vaccine of FMDV.