Roles of N-glycosylation in immunity of prME and NS1 gene of JEV / 病毒学报

PrME and NS1 gene were the two main immuneprotect proteins of Japanese encephalitis virus (JEV), and they were also N-linked glycosylation proteins. To clear the effect of N-glycosylation on JEV immunity, the N-glycosylation site of prME and NS1 gene were eliminated by site-directed mutant PCR, subtituting the N to Q. And the the mutant genes were subcloned into eukaryotic expression plasmid. Four-weeks female mice were immuned with the wildtype and mutant gene by twice. The antibodies against prME were detected by ELISA and the neutralization antibodies were tested by viral neutralizing assay. The immunoprotection were determined by attack with JEV virulent strain. Compare with the wild-type gene immuned-groups, one N-glycan eliminated prME gene could induce a little higher ELISA antibody, neutralization antibody and immunoprotection, but the immunity of gene with both N-glycan absence was decreased. The similar status were observed in the wildtype and mutant NS1 groups. Thus these results show that the N-linked glycosylation in the prME and NS1 gene were correlated with the immunity, one glycan absent would enhance the immunity but both two loss would impair it.

Study on the feasibility of a cell-culture inoculation test in the detection and isolation of street rabies virus / 中华流行病学杂志

Objective Feasibility of using MNA cell-culture inoculation test to detect and isolate the street rabies virus. Methods Using MNA cell-culture inoculation test, fluorescent antibody test (FAT) and sandwich ELISA with double-antibodies to detect 33 specimens of street rabies virus, 20 specimens of negative canine brains and 4 specimens of healthy mice brains. Results 33 specimens of street rabies virus were positive to the cell-culture inoculation test but the others were negative. The concordances of MNA cell-cultured inoculation test with FAT and sandwich ELISA with double-antibodies were both 100%. Conclusion MNA cell-culture inoculation test appeared to be both highly sensitive and specific in detecting the street rabies virus, and could be used in detection and isolation of the virus.

Construction of the recombinant adenovirus carrying porcine interferon gamma (poIFNgamma) and identification of its antiviral activity / 病毒学报

The total RNA was extracted from peripheral blood mononuclear cells (PBMC) which was isolated from Meishan porcine and induced with concanavaline A (ConA), then the porcine interferon gamma gene (PoIFNgamma, 501bp) was amplified by RT-PCR. The result of sequencing demonstrated that the amplified PoIFNgamma had 100% nucleotide homology with the other porcine IFNgamma sequence published on GenBank. The objective gene (PoIFNgamma) was inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNgamma. And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37 degrees C for 24h on kanamycin resistance plate and selected for smaller colonies. Then, the extracted recombinant plasmid was named pAd-Sh-PoIFNgamma, which was confirmed by Pac I digestion, and transformed into XL10-Glod(r) for copious preparation. pAd-Sh-PoIFNgamma linearized with Pac I was co-transfected with liposome into 293 package cell-line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome (deleted with E1 and E3 genes) carrying PoIFNgamma was successfully packaged into intact virion. The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR. The antiviral activity of PoIFNgamma was tested by CPE50 method. The results showed that the PoIFNgamma expressed by adenovirus had high antiviral activity, which was 1.3 x 10(6) U/mL against VSV in MDBK cells. The results demonstrated that the recombinant adenovirus carrying PoIFNgamma could be stably passaged.

Infectious bovine rhinotracheitis viral gG expression and gG-ELISA development / 生物工程学报

Taking the genome DNA of Infectious Bovine Rhinotracheitis Virus (IBRV) as the template, the gG gene was amplified with PCR and cloned into the T cloning vector pMD18-T. After being identified by restriction digestion and DNA sequencing, the insert was subcloned into the expression vector pGEX-KG. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot assay showed that this gene was expressed as both soluble form and inclusion body by the transformed E. coli BL21 strain (DE3). The fusion protein was purified and used as the coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay (ELISA). Comparison between this gG-ELISA and commercial IBRV gB-ELISA Kit (IDEXX) was made in the detection of 380 cow serum samples. The results demonstrated an agreement of 92%. By using this novel gG-ELISA, 1248 cow serum samples were tested and the average positive rate of IBRV antibodies for imported cows is 21.7%, while the positive rate ranged greatly from 0.0%-41.5% for Hubei local Chinese Black and White Dairy Cows.

Preparation antibodies against recombinant bovine IFN-gamma and development of sandwich ELISA for bovine IFN-gamma detection / 生物工程学报

This study was aimed to establish ELISA for recombinant bovine IFN-gamma (BovIFN-gamma) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-gamma (BovIFN-gamma) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-gamma. The pETBovlFN-gamma was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-gamma as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-gamma were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG1 . Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-gamma, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-gamma specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-gamma, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-gamma paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.

Study on immunogenicity of the N-terminal polypeptide of RTX toxin I of Actinobacillus pleuropneumoniae / 生物工程学报

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.

Acute toxicity and immunoprotection of recombinant apxI toxin of Actinobacillus pleuropneumoniae in mice / 生物工程学报

Acute toxicity and immunoprotection of Actinobacillus pleuropneumoniae (APP) ApxI toxin recombinant proteins (include crude inclusion bodies and refolded recombinant protein) were evaluated in mice, and compared with the natural ApxI extracted from culture supernatant of APP serotype 10. In the acute toxicity experiment, the three proteins were intraperitoneally injected into Kunming mice in a dose of 200microg per mouse. The body and organ weight, heamatological and biochemical indexes were examined at 24h, 7 days and 14 days post administration. There was no death after the intraperitoneal administration of the three proteins, and no significant change was found in the body weight, organ indexes, heamatological and biochemical indexes. To study their immunoprotection, the three proteins were emulsified with Freund's adjuvant respectively and vaccinated the mice twice with a 2-week of interval. Two weeks after the second vaccination, the mice were challenged intraperitoneally with a lethal dose of APP serotype 10 (1.09 x 10(8) cfu), and serums were examined by an ApxI-specific ELISA. The results revealed that the recombinant protein had a good immunogenicity and could induce protection immune reaction.

Construction and characterization of delta crp delta asd mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain / 生物工程学报

Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid, attenuated by chemical method. Although the vaccine has good immunogenicity, it remains some residual virulence. In order to develop a safer vaccine strain and exploit C500 as a live vaccine vector for mucosal immunization, delta crp delta asd double deletion mutant was constructed. Firstly, the recombination suicide vector with 320 bp-deleted crp (cAMP receptor protein) gene and sacB (sucrose-sensitive gene) gene was constructed and conjugated with C500. The unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR. Then the asd (beta-aspartic semialdehyde dehydrogenase) gene was further deleted in the delta crp strain by the same method. Foreign DAP (diaminopimelic acid) must be supplied for delta crp delta asd mutant to grow. The phenotype, growth properties and virulence in mice of delta crp mutant were further characterized. In conclusion, the delta crp delta asd double-deletion mutant was successfully constructed. The delta crp delta asd mutant can be used as a live vector to express foreign genes and to develop potential oral multivalent vaccines.

Cloning, prokaryotic expression of chicken interferon-alpha gene and study on antiviral effect of recombinant chicken interferon-alpha / 生物工程学报

The full length of chicken interferon alpha (ChIFN-alpha) gene was amplified by the polymerase chain reaction (PCR) from total liver genome of Sanhuang meat-chicken and sequenced. The amplified gene was about 582bp. The coding region for mature protein (489bp) was subcloned into pET-28a(+). The recombinant plasmid pET-28a(+)-IFNalpha was identified by enzyme digestion and DNA sequencing. Data of SDS-PAGE and Western-blot indicated that a 22kD fusion protein was expressed in the form of inclusion bodies with good immunity. The purity of inclusion bodies was above 70% and that of protein purified by nickel affinity chromatography was 95%. The recombinant protein could inhibit H9N2 avian influenza virus (H9N2 AIV) replication on chick embryo fibroblast. 2 microg of recombinant IFN-alpha could completely protect Chick embryo from H9N2 AIV infection. The recombinant IFN-alpha can also delay Newcastle disease virus (NDV) replication on chick embryo for 12 approximately 48h. Chicken administered recombinant IFN-alpha can resist the H9N2 AIV infection. The bioactivities of recombinant IFN-alpha purified by affinity chromatograph were 20 times higher than that of inclusion bodies.

High expression of the foot-and-mouth disease's structural protein P1 in Escherichia coli and analysis of its biology activity / 生物工程学报

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.

Immunogenicity of DNA vaccine expressing GP5 of porcine reproductive and respiratory syndrome virus fused with VP22 of bovine herpesvirus 1 / 生物工程学报

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus / 生物工程学报

Pseudorabies virus (PRV), an alpha-herpesvirus, has been used as a vector for live-viral animal vaccines. The recombinant PRV TK- / gE- / GP5+, which expressing GP5 of PRRSV, is developed based on the PRV genetic-depleting vaccine-virus strain, TK- / gE- /LacZ+. However, this strain stimulated poorly the vaccinated animals to produce neutralizing antibodies against PRRSV. In order to develop a booster specific immunized response of the PRV recombinant, the ORF5 gene of PRRSV TK- / gE- / LacZ+ was substituted by a modified ORF5 gene, ORF5m. The resultant recombinant PRV, TK- /gE- / GP5m+, was verified by PCR, Southern blotting and Western blotting. TK- / gE- / GP5m+ and TK- / gE- / GP5+ expressed GP5 proteins were inoculated into balb/c mice to evaluate their immunogenicity. The results demonstrated that the amount of neutralization antibodies and cell-immunity responses induced by TK- / gE- /GP5m+ against PRRSV were higher than that of TK- / gE- / GP5+. This study indicated that the new recombinant PRV expressing the modified GP5m protein is a candidate for the development of bivalent genetic engineering vaccines against PRRSV and PRV.

The expression of porcine circovirus type 2 ORF2 gene in insect cells and its character / 生物工程学报

To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter.

Expression of GP5-M fusion protein of porcine reproductive and respiratory syndrone virus (PRRSV) and establishment of ELISA diagnose based on the recombinant fusion protein / 生物工程学报

The cDNA fragment encoding the truncated GP5 and the full-length M protein of Porcine Reproductive and Respiratory Syndrone Virus (PRRSV) were orderly fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKG-56. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-GP5-M fusion protein was expressed in high level. Western-blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against PRRSV. The fusion protein was further purified and used as an antigen to establish a novel PRRSV ELISA diagnose assay (P56-ELISA). Comparison between P56-ELISA and the abroad kit IDEXX-ELISA showed the two methods had 94.1 percent agreement by detecting 205 serum samples, indicating that the indirect P56-ELISA was specific and sensitive. The correlation between virus neutralization antibody of the infected pigs (not convalescent pigs) and antibody response to the fusion protein GP5-M was further studied. The regression function analysis suggested that there was no significant correlation between ELISA antibody response (OD630 nm) to the fusion protein GP5-M in clinical serum and their specific neutralizing titers.

Expression of hemagglutinin of avian influenza virus (AIV) and its application in diagnosis of AIV H9 subtype / 生物工程学报

In order to differently diagnose avian influenza virus (AIV) subtypes, the HA gene of AIV H9 subtype was cloned, expressed and utilized in an enzyme-linked immunoad sorbent assay (ELISA). HA gene (1683bp) of H9N2 AIV was amplified by RT-PCR from a strain of field isolated H9N2 AIV, and its identity was confirmed by sequencing. The HA gene was subcloned into prokaryotic expression vector pGEX-KG with its secretion signal sequence removed. The expressed HA-GST fusion protein in E. coli BL21 was characterized by SDS-PAGE and western blotting analysis as a 90kD protein with immunogenicity. The fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation. The HA-GST fusion protein was used to establish an indirect ELISA for the detection of antibodies to H9 subtypes of AIV. The assay has 91.57% specificity to H9 AIV, 92.31% sensitivity and excellent reduplication. It could be used to differently detect antibodies to H9 AIV.

Recombination and expression of ORF1 and ORF2 gene of porcine circovirus type 2 and gene of pseudorabies virus / 生物工程学报

ORF1 and ORF2 gene of porcine circovirus type 2 were cloned by PCR with the specific primers designed according to genome of PCV2 (AY035820). Following extraction and digestion, PCR products were subsequently inserted into universal transfer vector plECMV (deleted partial gE and gI of pseudorabies virus) to generate recombinant transfer plasmid pIEORF1-ORF2. The genomic DNA of PRV TK-/gE- /LacZ+ strain and pIEORF1-ORF2 were co-transfected into IBRS-2 cells with lipofectin, and recombinant virus TK- /gE- /gI- /ORF1-ORF2+ was selected by PCR with ORF1 gene and ORF2 gene primers respectively. The recombinant virus was analyzed with Southern blotting and Western blotting. The results indicated that ORF1 and ORF2 gene of PCV2 had been inserted into the genome of TK- /gE- /LacZ+ strain and the expressed ORF1-ORF2 fusion protein could react with PCV2 positive sera. Result of virus titers detection showed the insertion of ORF1 and ORF2 gene did not influence propagation of recombinant virus.

Construction and characterization of a pseudorabies virus TK-/gG- mutant / 生物工程学报

To construct a TK-/gG- mutant of pseudorabies virus, the gG-detected transfer vector pUSKKBB and genomic DNA of pseudorabies virus TK-/gG-/LacZ+ were co-transfected into IBRS-2 cells. Transfection progeny were plated onto PK-15 cells and incubated for 2 days under methylcellulose. Then the overlay was removed and replaced by 1% low melting point agarose in DMEM supplemented with 150 microg/mL X-gal. After 2 days, white plaques were screened for and purified 4 times. By PCR amplification of gG-deleted gene and LacZ gene, a recombinant virus with TK-/gG- phenotype was confirmed. Sequence of the PCR product revealed that there were 1,176 bp detection in gG gene of the PRV TK-/gG- mutant. Amplifying the gG-deleted gene of different generations of the TK-/gG- mutant showed that the mutant was stable within PK-15 cells. TCID50 assay indicated that the recombinant virus grows well on PK-15 cells. The mice immunized with the TK-/gG- virus showed no sign of abnormality. As a control, all mice inoculated with PRV strain died from the infection. All mice that received TK-/gG- survived after a lethal PRV challenge. However none of the mice injected with phosphate-buffer saline (PBS) survived from the challenge. The above results demonstrated that the recombinant virus could be a candidate marker vaccine strain for eradicating pseudorabies in pig herds.

Cloning，Sequence Analysis and Expression in E.coli of the EP0 Gene of Pseudorabies Virus Ea Strain / 中国病毒学

The 1.23 kb DNA fragment encoding the early protein EP0 of pseudorabies virus (PRV) Ea strain was amplified by PCR technique and cloned into pBluescriptII sk+.Three sequencing plasmids containing the partial fragment of the EP0 gene were constructed and the sequences were obtained by Sanger's sequencing technique. Compared with PRV InFh strain, there were multipile site-mutations and a deleted-mutation in the EP0 gene of PRV strain Ea，and the diversity of amino acid residues also existed.Then, the EP0 gene was inserted into an expression vector, pET-28a, fused into the downstream of the 6ΧHis-Tag in frame, to yield the expression plasmid pETEP0. After induction by IPTG, a high expression of fusion protein was obtained, SDS-PAGE analysis and Western blotting showed that the fusion protein was 62kD and the protein was specific to antisera against PRV Ea strain. This indicated that the EP0 gene be expressed in BL21(DE3) and the expression products have immuno-genicity.