Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2 / 中国血吸虫病防治杂志

Objective To construct pEGFP-N1-HBsAg-ROP2 recombinant expression plasmid and transfect HEK293T cells for expression,and pay a way for Toxoplasma gondii nucleic acid vaccine development. Methods According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites,the HBsAg gene was amplified by PCR.The HB-sAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene.The HBsAg-ROP2 fragment was amplified by PCR and digested with HindⅢand KpnⅠto clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2.The expression vector was transfected into HEK293T cells based on the identification of PCR amplifi-cation,restriction endonucleases and sequencing.Results The PCR product of HBsAg was about 700 bp,which was consis-tent with the theoretical value.Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3-HBsAg-ROP2 recombinant plasmid.The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment.The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank.The target plasmid was successfully transfect-ed into HEK293T cells,and the expression was correct,the protein concentration was 3.08 mg/ml.Conclusion The recombi-nant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.

Immunogenicity of dominant epitopes of complex antigen rhoptry protein 2-sunface protein 1 derived from Toxoplasma gondii / 中华传染病杂志

Objective To analyze the immunogenicity of dominant epitope of complex antigen of rhoptry protein 2 (ROP2 ) and major surface protein 1 (SAG1 ) derived from Toxoplasma gondii (T .gondii) .Methods Dominant epitope of ROP2‐SAG1 containing both dominant T‐and B‐cell epitopes was predicted and selected from T . gondii with bioinformatics methods .The gene fragment cloned into pET32a expression vector was transformed into the competent cell (Escherichia coli strain Rosetta) and expressed under the induction .The protein purified by nitrilotriacetic acid (Ni‐NTA) agarose resin were finally identified by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis . Japanese rabbits were immunized subcutaneously with purified epitope protein in contrast with control group immunized with pET32a protein or phosphate buffevred saline (PBS) .The sera from immunized rabbits were collected at week 0 , week 2 and week 4 for determination of epitope‐specific antibody IgG using enzyme‐linked immunosorbent assay ,and immunodot assay was used to further confirm the specificity of antibody .After BALB/c mice were immunized with purified epitope proteins ,the capacity of production of interferon‐γ(IFN‐γ) in splenocytes was detected by enzyme‐linked immunospot assay .Results Relative molecular weight 30 000 of dominant epitope was derived from prokaryotic system .Then the rabbits immunized with purified dominant epitope could produce corresponding epitope‐specific antibody IgG . And with the increased frequency of immunization ,the level of antibody gradually increased .At week 2 and 4 ,higher antibody response were observed in group of rabbit immunized with dominant epitope than those of control group(1.454±0.098vs0.616±0.084,F=0.000,P<0.05;2.299±0.224vs1.580±0.192,F=0 .112 ,P< 0 .05) .The antibody titer at week 4 was as high as 1∶40 960 .Immunodot assay further confirmed the antibody specificity against the dominant epitope .The level of IFN‐γ in splenocytes from mice immunized with dominant epitope after stimulation with three epitope specific CTL peptides (epitope peptides 1 [19 .333 ± 1 .528]/100 000 cells ,epitope peptides 2 ([40 .333 ± 1 .528]/100 000 cells) ,epitope peptides 3 ([70 .667 ± 1 .890]/100 000 cells) was significantly higher than that of PBS control group (epitope peptides 1 [1 .033 ± 0 .150]/100 000 cells ,epitope peptides 2 [1 .045 ± 0 .110]/100 000 cells , epitope peptides 3 [1 .041 ± 0 .120]/100 000 cells , F=0 .284 ,0 .000 and 0 .284 ,respectively ;all P<0 .05) . The level of IFN‐γ from splenocytes stimulated with combined peptide with cytotoxic T lymphocyte (CTL) epitope peptide and helper T cell epitope peptide (epitope peptides 3) was significantly higher than that with single CTL epitope peptides (epitope peptides 1 and epitope peptides 2 , F=5 .796 and 0 .000 ,respectively ;both P<0 .05) .Conclusion Screened dominant epitopes of ROP2‐SAG1 from T .gondii derived from prokaryotic expression system exhibit remarkable immunogenicity .

Construction and identification of pcDNA3-HBsAg-p30-ROP2 expression vec-tor / 中国血吸虫病防治杂志

Objective To construct a multi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector and identify it preliminarily. Methods According to recombinant pcDNA3-p30-ROP2 restriction sites,HBV HBsAg gene sequences of primers were designed and synthesized to amplify target fragment,and then cloned into pcDNA3-HbsAg-p30-ROP2 expression vector. Af-ter sequencing,it was identified finally by restriction enzyme digestion and other molecular biology techniques. Results HBV HBsAg gene segment was amplified by PCR and the multi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector was constructed and identified to be correct as theoretical values. The PCR and restriction enzyme digestion results showed that HBsAg and p30-ROP2 gene in recombinant plasmid were confirmed by DNA sequencing. Conclusion The multi-gene recombinant pcD-NA3-HBsAg-p30-ROP2 expression vector is successfully constructed.

High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Expression and identification of the multiple gene ROP2-P30 of Toxoplasma gondii in E.coli BL21 / 中国人兽共患病学报

To obtain the functional fusion protein of rhoptry protein 2, compound rhoptry protein2 and surface antigen 1 of Toxoplasma gondii. the ROP2 and P30 genes from genomic DNA of T.gondii RH strain were amplified by PCR, and were inserted into pMD18-T cloning vector. Then the ROP2 fragment was subcloned to pET-30a(+) plasmid digested by EcoRⅠand Hind Ⅲ to construct plasmid pET-ROP2. Furthermore,the P30 fragment was subcloned into pET-ROP2 digested by BglⅡand EcoRⅠto create plasmid pET-ROP2-P30, the resulting recombinant plasmids , transformed into E.coli BL21 (DE3), were induced with IPTG. and the proteins identified by SDS-PAGE were further purified and refolded. The biological activity was analyzed by Western blot with specific antibody. It was found that the sizes of ROP2 and ROP2-P30 were 1212 and 1896bp with corresponding molecular weight 50- kDa and 75-kDa, respectively. The recombinant protein ROP2 (50-kDa) could specifically react with rabbit-polyclonal antiserum, and complex fusion protein ROP2-P30 (75- kDa) could react with P30 monoclonal antibody.

Cloning and Expression of the Fused Gene of Rhoptry Protein ROP2 and Major Surface Protein P30 from Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To clone and express the fused gene fragment coding rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii as a preparation for the construction of the complex ROP2,P30 antigen by gene engineering.Methods The gene fragment encoding P30 was amplified by PCR from T.gondii RH strain and subcloned into the recombinant plasmid pUC119/ROP2 already constructed.The recombinant plasmid pUC119/ROP2,P30 was digested by SacⅠ/HindⅢ and inserted into the same site of expression vector pET28b.The recombinant plasmid of pET28b/ROP2,P30 was transformed to E.coli and expressed under the induction of IPTG.Results The gene fragment 700 bp encoding P30 was obtained from the total DNA of T.gondii by PCR.The recombinant plasmid pET28b/ROP2,P30 was successfully constructed,which was highly expressed in E.coli,a fusion protein with molecular weight of 69 000.Conclusion The fusion gene encoding the rhoptry protein ROP2 and the major surface protein P30 of T.gondii has been successfully cloned and expressed to be an expected recombinant fusion protein ROP2,P30 with molecular weight 69 000.

Studies on the Immuno-Protection of ROP2 Nuclei Acid Vaccine in Toxoplasma gondii Infection / 中国寄生虫学与寄生虫病杂志

Objective To study the protective effect of ROP2 nuclei acid vaccine in mice.Methods Forty-two BALB/c mice were divided into three groups.Each mouse in experiment group was injected with 50 ?g recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris.In control groups,each mouse was injected with 50 ?g blank plasmid pc-DNA3 and with 50 ?l PBS respectively.All mice were immunized for three times with an interval of three weeks.The volume was doubled for the final injection in the two plasmid groups.Blood,spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4+,CD8+ T cells and cytokines 2 weeks after the final immunization.The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation.Results The vaccine induced strong cellular and humoral immune response.The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro.The lymphocyte phenotype was analyzed.CD4+ T cells proliferated sharply(69.5?3.4)%,and the ratio of CD4+/CD8+ increased considerably by(4.69?1.32)%(P