[Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2].

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 HBsAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene. The HBsAg-ROP2 fragment was amplified by PCR and digested with Hind Ⅲ and Kpn Ⅰ to 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 amplification, restriction endonucleases and sequencing. RESULTS: The PCR product of HBsAg was about 700 bp, which was consistent with the theoretical value. Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3HBsAg-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 transfected into HEK293T cells, and the expression was correct, the protein concentration was 3.08 mg/ml. CONCLUSIONS: The recombinant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.

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.

[Prokaryotic expression and identification of rhoptry protein 38 of Toxoplasma gondii].

OBJECTIVE: To explore the biological function of rhoptry protein 38 (ROP38) of Toxoplasma gondii, and to identify the reactogenicity of the recombinant protein (rROP38). METHODS: The ROP38 was amplified by RT-PCR from T. gondii RH strain, and was cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. Then the rROP38 was analyzed by SDS-PAGE and identified by Western blot. RESULTS: SDS-PAGE showed that rROP38 was efficient expression with a molecular weight of about 43 kD. Western blot showed that rROP38 reacted with antibody of His tag or human positive antibody, which indicated that ROP38 had good reactogenicity and could be a serological diagnostic antigen. CONCLUSIONS: The study successfully obtains the rROP38 of T. gondii with good reactogenicity.

Epidemiological Investigation of Asymptomatic Dogs with Leishmania Infection in Southwestern China Where Visceral Leishmaniasis is Intractable

Heishui county, located in northwest Sichuan province, southwestern China, is an endemic area of zoonotic visceral leishmaniasis (VL) and is the most intractable area. VL is never destroyed in it. Asymptomatic dogs (Leishmania parasites have been diagnosed but clinically healthy) are considered to be a potential reservoir host in zoonotic VL area, and most can lead to infection of individuals, that is a new challenge for controlling VL in humans. The present study aimed to assess the Leishmania infection rate of asymptomatic dogs in Heishui county. Total 105 asymptomatic domestic dogs were gathered from 4 districts in Heishui county to investigate the infection rate with serological and molecular methods based on ELISA and kinetoplast minicircle DNA(kDNA) PCR, respectively. Out of 105 dogs, 44 (41.9%) were positive by more than 1 method; 21 (20.0%) were positive by ELISA, and 30 (28.6%) were positive by kDNA-PCR. Our study showed that Leishmania infection of domestic dogs which is clinically healthy is prevalent in the studied district, and the asymptomatic dogs infected by Leishmania may be the primary reason for the prevalence of visceral leishmaniasis in the area.

Cloning and expression of gp85 gene of subgroup J avian leukosis virus isolated from hemangiomas / 病毒学报

During July to November in 2007, several outbreaks of Hemangiomas in Hy-line Brown laying hens were observed in China. The virus that infected these flocks was identified in cultured DF-1 cells by PCR and indirect fluorescent assay (IFA) with ALV-J specific monoclonal antibody JE-9. The gp85 gene of one strain named WS0705 of ALV-J was cloned and expressed. Phylogenetic analysis showed that gp85 amino sequences of WS0705 strain had the highest homology with that of the prototype HPRS-103. The gp85 gene from a constructed plasmid pMD18-T-WS0705gp85 was cloned into baculovirus transfer vector. rBac-WS0705gp85 was obtained by the Bac-to-Bac baculovirus expression system. The rBac-WS0705gp85 protein was analyzed by indirect immunofluor escence assay and Western blot. The results showed that positive green fluorescent was present in Sf9 cells infected with the recombinant virus and a 35 kD band was present in western blot. It is concluded that WS0705 gp85 gene was expressed in Sf9 cells infected with the recombinant virus and the SU protein of WS0705 can bind specifically to JE9 MAb of ALV-J. The expressed protein can be used to detect hemangiomas induced by ALV-J.

Isolation and identification of avian leukosis virus-B from layer chickens infected with avian leukosis virus-J / 病毒学报

Two strains of Avian leukosis virus subgroup B (ALV-B) were isolated for the first time in China Hy-line White on the cultured DF-1 cells which were inoculated tissue samples from by an ELISA assay, a histopathology examination and a PCR-based diagnosis. The results from the ELISA assay indicated that the positive rate of serum antibodies to ALV-B and ALV-J virus were 16.3% (15/92) and 13% (12/92), respectively. The histopathological examination indicated that two types of tumor cells existed at same focus in liver and spleen, which mainly were myelocytoma cells and lymphosarcoma cells. The PCR-based diagnosis were performed as follows: the cellular DNA was extracted from the inoculated DF-1 cells; the specific fragments of 1100 bp and 924 bp were obtained by a PCR system with the diagnostic primers of ALV-B and ALV-J; and the PCR results for ALV-A, MDV and REV were all negative. Then, the amplified fragments of the two ALV-B stains were partially sequenced and shown an identity of 92.8%,94.7% with the prototype strain of ALV-B (RSV Schmidt-ruppin B). The identities of two ALV-J strains with the prototype strain HPRS-103 at 96.9%, 91.5%; The identities of two ALV-J strains with the American prototype strain at 85.9%, 81.5%. Our study had shown that ALV-B was isolated for the first time from the ALV-J infected commercial layer flocks in China. It also indicated that the chance of genetic recombination among various subgroups of ALV was increased.