A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1,...A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1, then the recombinant plasmid pWRA was introduced into the attenuated Salmonella typhimurium SL3261. When used as a live vaccine and administered orally (po), intravenously (iv) or intraperitoneally (ip),the recombinant strain was able to live in vivo and elicit specific humoral and cellular immunity in BALB/c mice and rabbits. As oral immunization is safe and effective, it is thought that the live recombinant Salmonella tyPhimurium vaccine may bring the Pf oral live vaccine a step nearer.展开更多
AIM： To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS： A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plas...AIM： To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS： A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes （CTLs） of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS： HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1：97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector：target ratio of 20：1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION： pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.展开更多
190-kilodalton glycoprotein (P190) of Plasmodium falciparum,precursor of themajor surface protein of merozoites,is considered a promising candidate for blood stage malarialvaccine.Six primers were designed according t...190-kilodalton glycoprotein (P190) of Plasmodium falciparum,precursor of themajor surface protein of merozoites,is considered a promising candidate for blood stage malarialvaccine.Six primers were designed according to the sequence of MAD20 strain,with a GCclamp and BamH Ⅰ site at the 5’-end of each one,and a GC clamp and Xba Ⅰ site at the 3’-endof each one.The primers were synthesized by phosphoramidite approach (User’s Manual ofABI Company) and purified using HPLC.Three fragments in the second,third and fourth con-served regions of P190 gene of Plasrnodium falciparum FCC1/HN strain isolated from theblood of patients in Hainan Province of China were amplified by the polymerase chain reaction(PCR).The amplified fragments were suhcloned into pUC18 vectors and sequenced using thedideoxy chain termination method.All three regions of P190 gene of FCC1/HN strain also werehighly conservative as compared with P190 gene of MAD20 (Papua New Guinea isolate),K1(Thailand isolate),Wellcome (West Africa isolate) and CAMP (Malaysia) strains ofPlasmodium falciparum.The C at position 81 in the second conserved block of P190 gene ofFCC1/HN isolate was substituted by T,which did not change the amino acid determined by thecodon corresponding to the substitution.The genes sequenced were cloned into rpGEX-2T,aglutathione S-transferase gene fusion system for expression.展开更多
Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667)respectively, which encode amino acid residues of conserved region Ⅰ and Ⅴ on the P190 antigen were amplified by polymerase chain reaction f...Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667)respectively, which encode amino acid residues of conserved region Ⅰ and Ⅴ on the P190 antigen were amplified by polymerase chain reaction from genomic DNA in FCCl/HN strain of Plasmodium falciparum isolated from Hainan Province, China. It was found that there were five base substitutions in the P190CR V, in comparison with the nucleotide sequences of MAD20 strain. These two fragments sequenced were inserted into pGEX-2T plasmid. E. coli JM109 (DE3) were transformed with the recombinant plasmids and the parental plasmid. The results showed that the two fragments were expressed as high-level C-terminal fusions with glutathione S-transferase (GST). The fusion proteins were easily purified from bacterial lysates by affinity chromatography using glutathione Sepharose 4B.展开更多
A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was ...A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was synthesized chemically. This gene (named HGFC) was cloned and connected with another hybrid gene (HPFA) synthesized previously to make a bigger hybrid gene (HGFCAC). HGFC and HGFCAC were cloned in an expression vector pWR450-1 and transformed into E. Coli JM109. The engineered bacteria could express fusion proteins with molecular weights of 65 and 77 kDa after inducing with isopropylthio-β-D-galactoside (IPTG). The expression rate was about 35% of total bacterial proteins. The expressed products showed sepcific immunological reaction with rabbit antibodies against P. falciparum peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala (EENVEHDA)by Western blotting. The fusion proteins were pruified by precipitation with amonium sulfate. gel filtration and ion-exchange chromatography and the purity was 82%. The purified protein reacted specifically with mouse immune serum against falciparum blood stage antigens by dot enzyme-linked immunosorbent assay (dot-ELISA).The fusion protein was emulsified with Freund's complete adjuvant (FCA) and used to immunize rabbits. The immune serum can recognize P. falciparum erythrocytic stage antigens of Fcc-1/HN strain and Yunnan strain and had weak cross reaction with P. vivax,but had no reaction with P. cynomlogi and P. berghei antigens. The protective effect of the antibody was tested by in vitro inhibition test to cultured falciparum parasites. Preliminary results indicated that the immune sera could effectively reduce the invasion rates of the parasites to red blood cells and inhibit the growth of the in vitro cultured falciparum parasites. The inhibitory capacity of the immune sera to parasite invasion is enhanced as the amount of the sera increases and the incubation time of the sera with the parasites is prolonged.It was shown that after 72 h incubation at 20% concentration with the parasites, the serum can suppress the multiplication of P.falciparum growth in vitro to a level of 80%.The immune sera caused dispersion of the parasite cytoplasm,atrophy of parasites,agglutination of free merozoites and degeneration of schizonts.展开更多
To construct live recombinant vaccinia virus, The HGFSP gene encoding CSP, MSA1, MSA2, RESA,IL-1 and TT epitopes was inserted into the Eec RI and Burn HI sites of pSK plasmid. After digested with Eco RI,bluntly ended ...To construct live recombinant vaccinia virus, The HGFSP gene encoding CSP, MSA1, MSA2, RESA,IL-1 and TT epitopes was inserted into the Eec RI and Burn HI sites of pSK plasmid. After digested with Eco RI,bluntly ended by Klenow enzyme and digested with Sac I, the HGFSP gene was cloned into the Sma I and Sac I sites of the vaccinia virus insertion vector (pJ2--16). Recombinant plasmids were identified by gel electrophoresis,restriction enzyme and enzyme map. Results evidenced that HGFSP gene fragment was correctly inserted into the cloning site of hemagglutinin (HA) gene of the pJ2--16 vector. The recombinant plasmids were trans feeted into Cos--7 cells, which were infected with wild type of vaccinia virus Tiantan strain, by means of lipofectamine. Two recombinant vaccinia viruses (HA) were screened and cloned by chicken hemadorption test in BHK21 cells. Indirect immunofluorescence assay (IFA), Dot--ELISA and Western blot with the antibodies against HGFSP protein expressed by E. colt showed that one of the 2 recombinant vaccinia virus expressed desired proteins in infected BHK21 cells. Western blot also showed that the molecular weight of 2 of expressed protein bands was about 23 kDa, according to the theoretical molecular weight of HGFSP protein. Further identification of immunological characters of recombinant virus is under way.展开更多
Objective:To analyse the rar gene repertoire and characterise the rhondroitin sulphate A (CSA)-binding activity of the Duffy-binding like(I)BI.) domains encoded by the var2csa gene of a Plasmodium falciparum(P.falcipa...Objective:To analyse the rar gene repertoire and characterise the rhondroitin sulphate A (CSA)-binding activity of the Duffy-binding like(I)BI.) domains encoded by the var2csa gene of a Plasmodium falciparum(P.falciparum) isolate in Hainan Province,China.Methods:The sequences of var DBL1 regions were PCR-amplified,sequenced and the sequence characteristics was bioinformalically analysed.Recombinant proteins encoded by the var2csa genes were expressed and purified.The binding activities of the recombinant proteins to CSA receptor was detected by ELISA assays.Results:Fifty six unique DBI.a sequences were obtained,and the sequences represented similar diversity to the var genes of the genome parasite 3D7.There are two var2csa genes in the P.falciparum isolated from Hainan Province.Unlike in other falciparum parasites such as HB3,the two var2csa genes are more diverged.The receptor-binding capacity of DBL-5εand DBI.-6 e domains of HN var2CSA was studied.Conclusions:This work represented the diversity of rar genes of a P.falciparum isolate in China.展开更多
Objective Four B and Th cell epitopes were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigeni...Objective Four B and Th cell epitopes were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigenicities were studied Methods The partially synthesized oligonucleotide was annealed, PCR amplified and cloned into a mammalian cell expression vector By using a pair of isocaudamers on the vector, different single copies of B epitopes were multiplied and were tenderly stringed into two groups of chimeric DNA vaccine with different configurations BALB/c mice were immunized with these DNA plasmids by either intramuscular or intradermal injections Results The antisera from the immunized mice tested by ELISA showed that only the configuration which had a single copy of universal T helper cell epitope, CS T3, located at the C terminal of the multi copy B cell epitopes induced a high antibody response The T helper cell epitope at any other position of the peptide, or the double T helper cell epitopes configured with the B cell epitopes did not enhance antibody response, and some configurations even decreased the humoral response to a B cell epitope Conclusion This study demonstrated that both combination and configuration of the epitope may affect the antigenicity of a chimeric multiple antigen展开更多
文摘A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1, then the recombinant plasmid pWRA was introduced into the attenuated Salmonella typhimurium SL3261. When used as a live vaccine and administered orally (po), intravenously (iv) or intraperitoneally (ip),the recombinant strain was able to live in vivo and elicit specific humoral and cellular immunity in BALB/c mice and rabbits. As oral immunization is safe and effective, it is thought that the live recombinant Salmonella tyPhimurium vaccine may bring the Pf oral live vaccine a step nearer.
基金Supported by the 135 Project of Jiangsu Province, No. 044
文摘AIM： To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS： A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes （CTLs） of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS： HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1：97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector：target ratio of 20：1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION： pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.
基金This study was supported by UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases(TDR),ID No.910265
文摘190-kilodalton glycoprotein (P190) of Plasmodium falciparum,precursor of themajor surface protein of merozoites,is considered a promising candidate for blood stage malarialvaccine.Six primers were designed according to the sequence of MAD20 strain,with a GCclamp and BamH Ⅰ site at the 5’-end of each one,and a GC clamp and Xba Ⅰ site at the 3’-endof each one.The primers were synthesized by phosphoramidite approach (User’s Manual ofABI Company) and purified using HPLC.Three fragments in the second,third and fourth con-served regions of P190 gene of Plasrnodium falciparum FCC1/HN strain isolated from theblood of patients in Hainan Province of China were amplified by the polymerase chain reaction(PCR).The amplified fragments were suhcloned into pUC18 vectors and sequenced using thedideoxy chain termination method.All three regions of P190 gene of FCC1/HN strain also werehighly conservative as compared with P190 gene of MAD20 (Papua New Guinea isolate),K1(Thailand isolate),Wellcome (West Africa isolate) and CAMP (Malaysia) strains ofPlasmodium falciparum.The C at position 81 in the second conserved block of P190 gene ofFCC1/HN isolate was substituted by T,which did not change the amino acid determined by thecodon corresponding to the substitution.The genes sequenced were cloned into rpGEX-2T,aglutathione S-transferase gene fusion system for expression.
文摘Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667)respectively, which encode amino acid residues of conserved region Ⅰ and Ⅴ on the P190 antigen were amplified by polymerase chain reaction from genomic DNA in FCCl/HN strain of Plasmodium falciparum isolated from Hainan Province, China. It was found that there were five base substitutions in the P190CR V, in comparison with the nucleotide sequences of MAD20 strain. These two fragments sequenced were inserted into pGEX-2T plasmid. E. coli JM109 (DE3) were transformed with the recombinant plasmids and the parental plasmid. The results showed that the two fragments were expressed as high-level C-terminal fusions with glutathione S-transferase (GST). The fusion proteins were easily purified from bacterial lysates by affinity chromatography using glutathione Sepharose 4B.
文摘A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was synthesized chemically. This gene (named HGFC) was cloned and connected with another hybrid gene (HPFA) synthesized previously to make a bigger hybrid gene (HGFCAC). HGFC and HGFCAC were cloned in an expression vector pWR450-1 and transformed into E. Coli JM109. The engineered bacteria could express fusion proteins with molecular weights of 65 and 77 kDa after inducing with isopropylthio-β-D-galactoside (IPTG). The expression rate was about 35% of total bacterial proteins. The expressed products showed sepcific immunological reaction with rabbit antibodies against P. falciparum peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala (EENVEHDA)by Western blotting. The fusion proteins were pruified by precipitation with amonium sulfate. gel filtration and ion-exchange chromatography and the purity was 82%. The purified protein reacted specifically with mouse immune serum against falciparum blood stage antigens by dot enzyme-linked immunosorbent assay (dot-ELISA).The fusion protein was emulsified with Freund's complete adjuvant (FCA) and used to immunize rabbits. The immune serum can recognize P. falciparum erythrocytic stage antigens of Fcc-1/HN strain and Yunnan strain and had weak cross reaction with P. vivax,but had no reaction with P. cynomlogi and P. berghei antigens. The protective effect of the antibody was tested by in vitro inhibition test to cultured falciparum parasites. Preliminary results indicated that the immune sera could effectively reduce the invasion rates of the parasites to red blood cells and inhibit the growth of the in vitro cultured falciparum parasites. The inhibitory capacity of the immune sera to parasite invasion is enhanced as the amount of the sera increases and the incubation time of the sera with the parasites is prolonged.It was shown that after 72 h incubation at 20% concentration with the parasites, the serum can suppress the multiplication of P.falciparum growth in vitro to a level of 80%.The immune sera caused dispersion of the parasite cytoplasm,atrophy of parasites,agglutination of free merozoites and degeneration of schizonts.
文摘To construct live recombinant vaccinia virus, The HGFSP gene encoding CSP, MSA1, MSA2, RESA,IL-1 and TT epitopes was inserted into the Eec RI and Burn HI sites of pSK plasmid. After digested with Eco RI,bluntly ended by Klenow enzyme and digested with Sac I, the HGFSP gene was cloned into the Sma I and Sac I sites of the vaccinia virus insertion vector (pJ2--16). Recombinant plasmids were identified by gel electrophoresis,restriction enzyme and enzyme map. Results evidenced that HGFSP gene fragment was correctly inserted into the cloning site of hemagglutinin (HA) gene of the pJ2--16 vector. The recombinant plasmids were trans feeted into Cos--7 cells, which were infected with wild type of vaccinia virus Tiantan strain, by means of lipofectamine. Two recombinant vaccinia viruses (HA) were screened and cloned by chicken hemadorption test in BHK21 cells. Indirect immunofluorescence assay (IFA), Dot--ELISA and Western blot with the antibodies against HGFSP protein expressed by E. colt showed that one of the 2 recombinant vaccinia virus expressed desired proteins in infected BHK21 cells. Western blot also showed that the molecular weight of 2 of expressed protein bands was about 23 kDa, according to the theoretical molecular weight of HGFSP protein. Further identification of immunological characters of recombinant virus is under way.
基金supports to Q.Chen from the National Basic Research Program of China(973 Program,No.2007CB513100)national science and technology project(2008ZX-10004-011)NSFC grant to J.Yin (30771886),N.Jiang(81171592,)and Q.Chen(81130033)
文摘Objective:To analyse the rar gene repertoire and characterise the rhondroitin sulphate A (CSA)-binding activity of the Duffy-binding like(I)BI.) domains encoded by the var2csa gene of a Plasmodium falciparum(P.falciparum) isolate in Hainan Province,China.Methods:The sequences of var DBL1 regions were PCR-amplified,sequenced and the sequence characteristics was bioinformalically analysed.Recombinant proteins encoded by the var2csa genes were expressed and purified.The binding activities of the recombinant proteins to CSA receptor was detected by ELISA assays.Results:Fifty six unique DBI.a sequences were obtained,and the sequences represented similar diversity to the var genes of the genome parasite 3D7.There are two var2csa genes in the P.falciparum isolated from Hainan Province.Unlike in other falciparum parasites such as HB3,the two var2csa genes are more diverged.The receptor-binding capacity of DBL-5εand DBI.-6 e domains of HN var2CSA was studied.Conclusions:This work represented the diversity of rar genes of a P.falciparum isolate in China.
文摘Objective Four B and Th cell epitopes were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigenicities were studied Methods The partially synthesized oligonucleotide was annealed, PCR amplified and cloned into a mammalian cell expression vector By using a pair of isocaudamers on the vector, different single copies of B epitopes were multiplied and were tenderly stringed into two groups of chimeric DNA vaccine with different configurations BALB/c mice were immunized with these DNA plasmids by either intramuscular or intradermal injections Results The antisera from the immunized mice tested by ELISA showed that only the configuration which had a single copy of universal T helper cell epitope, CS T3, located at the C terminal of the multi copy B cell epitopes induced a high antibody response The T helper cell epitope at any other position of the peptide, or the double T helper cell epitopes configured with the B cell epitopes did not enhance antibody response, and some configurations even decreased the humoral response to a B cell epitope Conclusion This study demonstrated that both combination and configuration of the epitope may affect the antigenicity of a chimeric multiple antigen
