Comparison of the antigenicity of African swine fever virus p35 protein as diagnostic antigen / 生物工程学报

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.

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.

Construction and identification of prokaryotic expression system for galactose/N-acetyl galactosamine-specifric lectin p30 gene from Cryptosporidium / 中国人兽共患病学报

To clone and construct a prokaryotic expression system containing the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium, the p30 gene was amplified from genomic DNA of Cryptosporidium parvum by PCR and cloned into vector pMD18-T directly. The positive clones were identified by EcoR I, Xho I digestion and sequenced. The gene structure and its possible function were analyzed and predicted by using related bioinformatics softwares. The P30 gene was recombined with plamid pET-28 a (+) to construct the prokaryotic expression vector and was expressed in E. Coli with IPTG induction. Ni-NTA affinity chromatography was used to extract P30 protein and the expression effect and purification of P30 protein were determined by SDS-PAGE and Western blotting. It was demonstrated that the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum was specifically amplified, and its sequence homology of nucleotide and the deduced amino acid sequence of P30 gene with relevant sequences in GenBank were 98%-100% and 99%-100% respectively. Its theoretical iso-electric point and molecular weight were found to be 6.4854 and 31842 dalton.It was predicted to contain 9 potential epitopes. The expressed plasmid was identified by EcoR I/ Xho I digestion and sequenced and the recombinant P30 protein could be identified by SDS-PAGE and Western blotting assay. It is evident that the prokaryotic expression system for galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum has been constructed successfully.

Detection and identification of TMV infecting tomato under protected cultivation in Paraná State

During an inspection in plastic houses in Sapopema, Paraná, 90 percent of tomato plants showed leaf abnormalities, probably associated with herbicide toxity. However, virus like symptoms developed in selected hosts after mechanical inoculatation. RT-PCR reactions using primers for an internal region within the movement protein gene of TMV and ToMV resulted in the amplification of a 409 bp cDNA fragment only by TMV primers. Deduced amino acids showed 100 percent identity when compared to TMV movement protein and 94 percent with ToMV. The RT-PCR protocol was efficient for quick and conclusive determination of virus species. The virus was purified and a polyclonal antiserum was raised for future surveys in tomato crops of Paraná. The partial genomic sequence obtained for TMV-Sapopema has been deposited under the accession number DQ173945, which is the first partial genomic sequence of an isolate of TMV from Brazil in the GenBank, and the first tomato virus isolate from Paraná to have some of its biological and molecular properties determined.

Durante uma inspeção em cultivos protegidos de tomate em Sapopema, Paraná, foram observadas anormalidades foliares em 90 por cento das plantas, indicando possivelmente a existência de um problema de fitotoxidade causada por herbicidas. Todavia, os sintomas manifestados nas hospedeiras após os ensaios de inoculação mecânica revelaram que os sintomas estariam relacionados a uma infecção por Tobamovirus. As reações de RT-PCR com oligonucleotídeos específicos para uma região interna da proteína de movimento de dois vírus comuns em tomate, TMV e ToMV, resultaram na amplificação de um fragmento de 409 pares de bases, apenas com os oligonucleotídeos específicos para o TMV. Após o sequenciamento, os aminoácidos deduzidos apresentaram identidade de 100 por cento quando comparados com as seqüências das proteínas de movimento de outros isolados do TMV, e 94 por cento de identidade com seqüências do ToMV. A RT-PCR demonstrou ser um método eficiente para a rápida e conclusiva determinação da espécie viral envolvida na infecção do tomateiro. A seqüência parcial do genoma do isolado de TMV de Sapopema, está depositada no GenBank sob o número de acesso DQ173945, sendo esta a primeira seqüência genômica parcial de um isolado de TMV do Brasil, e conforme o nosso conhecimento, o primeiro isolado de TMV do Paraná a ter algumas de suas propriedades biológicas e moleculares determinadas. Um anti-soro policlonal foi produzido, o que permitirá futuros levantamentos da ocorrência de Tobamovirus nas principais áreas de cultivo de tomate do Paraná.

Primary structure of mature SAG1 gene of an Indonesian Toxoplasma gondii and comparison with other strains

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.

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.

Over-expression and purification of the recombinant p30 antigen of Toxoplasma gondii / 中国人兽共患病学报

To provide the basis for preparation of diagnostic kits and vaccines in Toxoplasma gondii infection, the gene coding for the qualified recombinant p30 protein (SAG1) of this parasite was amplified by PCR, and the amplified gene was cloned into prokaryotic expression vector pET-30a(+) to construct the recombinant plasmid, and then transformed to E.coli DH5α. The positive recombinant plasmid was screened by PCR and double enzymes digestion, and the nucleotide sequence of p30 gene was determined by automated DNA sequencing. Meanwhile, the identified recombinant plasmid was transformed to E.coli BL21(DE3) with the expression of p30 on bacteria induced by IPTG and the expressed protein was identified by SDS-PAGE. The protein obtained was then further purified and refolded, and its biological activity was checked by Western blotting. It was shown that the size of the amplified gene was 750 bp with molecular weight of 30 ku, and this protein could specifically react with monoclonal antibody against p30 protein.

AMPLIFICATION, CLONING AND PROKARYOTIC EXPRESSION OF GENE FOR MAJOR SURFACE ANTIGEN OF TOXOPLASMA GONDII / 中国人兽共患病学报

Aim To amplify P30 gene and express P30 fusion with GST Methods P30 gene was smplified from T. gondii chromosomal DNA and ligated to pGEM-T and pGEX-4T-1. Screening-positive recombinants were induced for expression, which was subsequently detected by WB Results P30 gene was amplified and GST-fusion was confirmed by rabbit antiT. gondii serum. Conclusions The construction of pGEM-T-P30 and pGEX-4T-1-P30, together with the recombinant protein would lay a base for further investigation of P30 at a molecule-level and application to diagnosis and vaccination

The Construction of Recombinant Plasmids of Toxoplasma gondii P30 and the Immunoprotective Activity of the P30 Constructs / 中国寄生虫学与寄生虫病杂志

Objective To identify the potential DNA vaccine candidate which can induce the protective immune response to Toxoplasma gondii by inoculating mice with plasmid DNAs encoding three different forms of P30 antigen (membranous secretory,and intracellular). Methods Three forms of recombinant plasmid: pcDNA3 P30Mb(contain the whole P30 gene sequence,including the gene encoding signal peptide and hydrophobic tail),pcDNA3 P30Se(contain the whole P30 gene sequence, without the gene encoding hydrophobic tail) and pcDNA3 P30In(contain the whole P30 gene sequence,without the gene encoding signal peptide) were constructed by PCR and subcloning technique. The mice were immunized with different forms of recombinant plasmids and IgG antibodies in the mice were detected by ELISA and Western blotting. Results Three forms of expression recombinant plasmid of Toxoplasma gondii P30 gene were successfully constructed. The P30 inserts were identified by restrictive enzyme digestion and sequencing. ELISA and Western blotting analysis demonstrated that specific IgG antibody could be induced in three immunized groups, but there was some difference in appearence time and intensity of IgG.Conclusion Genetically immunization of mice with the recombinant plasmids could elicit specific IgG antibodies. In respect to IgG response, the immune efficiency of the three forms of recombinant plasmids was different at the beginning (2 wk),but 4 wk later approximately same.

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.

Expression and identification of P30 surface antigen from RH strain of Toxoplasma gondii / 中国血吸虫病防治杂志

Objective To express P30 surface antigen of RH strain of Toxoplasma gondii in E.coli BL21(DE3). Methods The P30 gene from Toxoplasma gondii was cloned to the pET28b vector after PCR, and the recombinant expression plasmid pET28b-P30 was constructed. Then the recombinants were transformed into E.coli BL21(DE3) after identified by the restriction enzyme digestion, PCR and DNA sequence determination annlysis. A single colony of E.coli BL21(DE3) containing the recombinant plasmid, pET28b-P30 was inoculated in LB culture, then diluted 1∶100 into 2 ml LB culture and induced by 0.2 mmol/L IPTG, and the expression product was identified by SDS-PAGE and Western blot. Results The recombinant plasmid of pET28b-P30 was constructed. ② Plasmid pET28b-P30 could express a specific 30 kDa fusion protein in E.coli BL21(DE3). Conclusions The expression plasmid which contains the gene fragment encoding P30 surface antigen of Toxoplasma gondii has been successfully constructed and is highly expressed in E.coli BL21(DE3) as an inclusion body.

Protective immunity effects of co-immunization with P30 DNA vaccine and protein vaccine / 中国血吸虫病防治杂志

Objective To study protective immunity effects of co-immunization with P30 DNA vaccine and protein vaccine. Methods Forty-eight 5-6 weeks old BALB/c female mice were divided into four groups (A,B,C,D), 12 mice of each group. In group A (control group) each mouse was immunized with 100 ?g pcDNA3.1 plasmid DNA by intramuscular (i.m.) for three times at week 0,2 and 4; in group B (P30 protein group) each mouse was immunized (i.m.) with 50 ?g rP30+50 ?g CFA for three times at week 0, 2 and 4; in group C (pcDNA3.1-P30 group) each mouse was immunized with 100 ?g pcDNA3.1-P30 plasmid DNA (i.m.) for three times at week 0, 2 and 4; in group D (P30 DNA+rP30 co-immunization group) each mouse was immunized with 100 ?g pcDNA3.1-P30 plasmid DNA (i.m.) for two times at week 0, 2 and immunized by subcutaneous with 50 ?g rP30+50 ?g CFA at week 4. Each mouse was infected with 100 tachyzoites of Toxoplasma gondii RH strain four weeks later after last immunization. The anti-P30 antibodies were detected with ELISA before the challenge. Results The P30 DNA vaccine was successfully constructed. High titers of anti-P30 antibodies were induced in each mouse immunized with DNA vaccine. The protective trial proved that there was no significant difference between control group and experimental group though the survival time of mouse from experimental group had been prolonged. Conclusion The P30 DNA vaccine could induced high titers of anti- P30 antibodies in immunized mice, and it may be a potential DNA vaccine candidate.

Cloning, Expression and Purification of the Major Surface Antigen P30 of Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To obtain protein of the major surface antigen P30 of Toxoplasma gondii by molecularcloning. Methods The gene of P30 (containing the whole P30 gene sequence, without the gene encoding signalpeptide) was obtained by polymerase chain reaction (PCR) using the primer designed according to the DNA sequence ofP30. The recombinant plasmid was constructed using EcoR Ⅰ, Xho Ⅰ and was then transformed into E. coli Top10. Thepositive clones were identified by restriction enzymes and DNA sequence analysis. The fusion protein was induced by IPTGand purified by affinity chromatography using ProBond~(TM) Resin (a kind of nickel-charged sepharose resin) and was identi-fied by SDS-PAGE and Western blotting. Results The products of PCR, cleavage and link reaction were same as ex-pected and the sequence of inserted fragment in the recombinant plasmid was same as reported except one synonymy muta-tion. A 58 kDa fusion protein was induced by IPTG and was purified by chromatography. Conclusion Fusion proteincontaining Toxoplasma gondii P30 was achieved and was provided as experiment material for further research.