Construction and Expression of a TRAP/CSP Chimeric Protein of Plasmodium falciparum / 中国寄生虫学与寄生虫病杂志

Objective To construct a chimeric protein of Plasmodium falciparum pre erythrocyte stage (named as PfCP 4). Methods Thrombospondin related anonymous protein (TRAP) and circumsporozoite protein (CSP) of Plasmodium falciparum have been considered important candidates for pre erythrocytic malaria vaccine. The sequences of ectodomain of TRAP (aa: 26-330) and (NANP) 19 repeat region and entire carboxy terminus of CSP were fused to generate the PfCP 4 via a hinge consisting of Gly Pro Gly. The 1577 bp sequence of PfCP 4 was synthesized by asymmetric PCR based method and the synthetic gene was inserted into pQE. The resulting plasmid was transformed into E. coli SG13009 for inducible expression with IPTG. The expression product was detected by Western blotting. Results The result of Western blotting showed that the entire PfCP 4 recombinant protein was produced under IPTG induction whereas no product was detected in the cell without induction. The molecule weight of the protein was 57 kDa which was identical to the expected size, and the product was recognized by polyclonal antibodies against CSP protein. Conclusion A chimeric protein of Plasmodium falciparum pre erythrocyte stage (named as PfCP 4) was constructed successfully.

Research and Development of Malaria Vaccine in China / 中国寄生虫学与寄生虫病杂志

It remains an urgent need to develop effective malaria vaccine for global control of malaria.Application of high technologies such as biotechnology has facilitated the process of vaccine development for malaria.In the past 30 years,a large number of vaccine candidate antigens for malaria have been identified and some of them are currently in clinical trials.Major progress in malaria vaccine development has also been made in China.The PfCP-2.9 blood stage vaccine for malaria has entered clinical studies and some other vaccine candidates including combination malaria vaccine are currently in pre-clinical studies.The availability of various national research programs and international funding has stimulated laboratory and pre-clinical studies of malaria vaccine candidates.It remains a long-term goal to develop a safe and effective malaria vaccine to control and even eliminate the disease in the world,and many issues including malaria immunology and various types of technologies need to be addressed.However,efforts need to be continued toward the goal.

Construction and Expression of Plasmodium berghei Chimeric Protein in Pichia pastoris and its Immunogenicity in Mice / 中国寄生虫学与寄生虫病杂志

Objective To produce an erythrocytic stage chimeric protein of Plasmodium berghei in Pichia pastoris and evaluate its immunogenicity. Methods The DNA sequences of AMA1 (Ⅲ) and MSP1-19 from P. berghei homologous to the corresponding sequences of P. falciparum chimeric antigen 2.9 (PfCP-2.9) were fused to generate a chimeric gene, designated as PbCP-2.9. The resulting gene was redesigned using Pichia preferential coden usage and expressed in P. pastoris in the secreted form. The recombinant protein was purified by Ni-NTA affinity chromatography. Three groups each with 10 BALB/c mice were im- munized subcutaneously with 20 ?g of purified PbCP-2.9 antigen formulated in Freund’s adjuvant, Montanide ISA720 and Montanide IMS 1 312, respectively. Three control groups each with 10 mice received only adjuvants emulsified with PBS. All the mice received three immunizations at 2-week intervals with the same dose of antigen. Serum samples were collected at preimmunization and one week after each immunization, and were analyzed for specific antibodies by ELISA and reaction with natural P. berghei proteins by IFAT. Results The PbCP-2.9 antigen with Mr 26 400 was successfully expressed in P. pastoris in secret- ed form. The recombinant protein can be recognized by the serum against blood stage parasites of P. berghei. High antibody responses were detected in all three PbCP-2.9-immune groups of mice by ELISA. However, mice immunized with PbCP-2.9 antigen in Freund’s adjuvant produced higher antibody titers than those with PbCP-2.9 antigen in Montanide ISA 206 and Montanide IMS 1312 adjuvants. The mean antibody titer in Freund’s adjuvant was 6.9-fold higher than in Montanide ISA 206 adjuvant and 5.6-fold higher than in Montanide IMS 1312 adjuvant after the second immunization (F=81.06, P

Determination of Free Thiols in the Chimeric Protein PfCP-2.9 of Plasmodium faldparum / 中国寄生虫学与寄生虫病杂志

Objective To determine the free thiols in the chimeric protein PfCP-2. 9 of Plasmodium falciparum expressed by Pichia pastoris. Methods Two experiments of reverse phase HPLC and Ellman' s reaction were applied to the PfCP-2.9 for the determination of its free thiols. For RP-HPLC analysis, three kinds of samples were tested: PfCP-2. 9, dithiothreitol-reduced PfCP-2.9 and indoacetic acid-alkylated PfCP-2.9. Results Both experiments showed that there were no any free thiols present in the PfCP-2. 9. Conclution The disulfide bonds between cysteine residues of PfCP-2. 9 were formed completely.

Genotyping and Polymorphism Analysis of Plasmodium falciparum Isolates by Nest-PCR and PCR-RFLP / 中国寄生虫学与寄生虫病杂志

Objective To analyze the efficiency and specificity of MSP2 alleles genotyping for Plasmodium falci-parum isolates by Nest-PCR and PCR-RFLP. Methods MSP2 alleles from Plasmodium falciparum isolates of Yunnan and Hainan were genotyped by Nest-PCR and PCR-RFLP, respectively, and the efficiency and specificity of the two me-thods were analyzed. Results The conventional Nest-PCR method could detect 79.8% (166/208) alleles of MSP2,and 65.7% (65/99) for 3D7 family, but could not identify the type of any allele. While PCR-RFLP showed 25.3% higher genotyping efficiency than Nest-PCR. Moreover, this method could identify the allele types. Conclusion PCR-RFLP genotyping technique is more efficient and specific than conventional Nest-PCR, and it is a convenient tool in the study on molecular epidemiology of malaria.

Regulation Mechanism of Variation in Plasmodium falciparum Var Gene Family / 中国寄生虫学与寄生虫病杂志

This is a review on the new progress in the study of regulation mechanism of Plasmodium falciparum Var gene family. The mutually exclusive expression system caused expression only one in 60 var genes while others were silenced. It was regulated on the transcriptional level mainly through three pathways:non-coding DNA elements,chromatin structure and perinuclear localization.

Expression and Immunogenicity Evaluation of Ectodomain and Subdomains of Plasmodium berghei Apical Membrane Antigen 1 / 中国寄生虫学与寄生虫病杂志

Objective To express and evaluate the immunogenicity of ectodomain and its subdomains of Plasmodium berghei apical membrane antigen 1(PbAMA-1).Methods Sequence of PbAMA-1 gene was isolated from the genome of P.berghei,and was redesigned and divided into three overlapped fragments according to its subdomain structure.The codon-optimized DNA fragments of PbAMA-1 were synthesized and inserted into vector pET32a for expression in E.coli and the recombinant proteins were purified by Ni-NTA column,followed by refolding in vitro.Mice and rabbits were immu-nized with the recombinant proteins formulated with Freund adjuvant.Titer of the specific antibodies was detected by ELISA and IFA.The immunized mice were challenged by P.berghei to evaluate protective efficacy in vivo.Results The sequence of the PbAMA-1 gene was shown to be identical to that published before.PbAMA-1 sequence was redesigned via codon optimization and synthesized.Both ectodomain and its subdomains of PbAMA-1 were successfully expressed in E.coli after induction.The proteins were isolated with the purity of more than 90% after Ni column purification and refolding in vitro.Immunization of mice with the recombinant proteins induced high level of specific antibodies.The antibody titer to ectodomain E after the 3rd immunization showed a strong immunogenicity at(34.4?0.15)?10-4.The antibodies interacted with the parasites by indirect fluorescence.The immunized mice were partially protected from the challenge of P.berghei.Conclusion The recombinant PbAMA-1 is highly immunogenic and induces protective immunity against the challenge of P.berghei.

Preparation and Characterization of Monoclonal Antibody Specific to PfCP-2.9 Chimeric Protein of Plasmodium falciparum / 中国寄生虫学与寄生虫病杂志

Objective To prepare and characterize monoclonal antibody against a malaria vaccine candidate, PfCP-2.9 chimeric protein of Plasmodium falciparum. Methods BALB/c mice were immunized with PfCP-2.9,and the spleen cells were used for fusion with SP2/0 cells. The monoclonal antibodies were analyzed by ELISA,Western blotting as well as growth inhibition assay. Result A monoclonal antibody was obtained. It interacted with the PfCP-2.9 recombinant protein by ELISA and Western blotting. The interaction of the monoclonal antibody with the protein was reduction-sensitive,indicating that the antibody recognized a conformational epitope. Moreover,the antibody also recognized the cultured parasites of P.falciparum by indirect immunofluorescent antibody test(IFA). When tested by growth inhibition assay,the antibody significantly inhibited parasite growth in vitro of 56% inhibition rate at the antibody concentration of 0.3 mg/ml. Conclusion A monoclonal antibody against PfCP-2.9 malaria vaccine candidate has been obtained,which recognizes a conformational epitope of the protein and natural protein.

CTL Response to Pre-erythrocytic Stage Vaccine Candidate of Plasmodium falciparum in HLA-A*0201 Transgenic Mice Detected by ELISPOT Assay / 中国寄生虫学与寄生虫病杂志

The importance of cytotoxic T-lymphocyte(CTL)against malaria parasite in pre-erythrocytic stage has been presented in relevant researches. In order to investigate whether one CTL epitope(YLNKIQNSL)involved in a chimeric pre-erythrocytic stage vaccine candidate of Plasmodium falciparum which was expressed and purified in the laboratory can stimulate in vivo CTL response,HLA-A*0201 transgenic mice were immunized with this vaccine candidate. Enzyme-linked immunosorbent spot(ELISPOT)assay was performed on the splenocytes from the immunized transgenic mice. Positive result indicated that this CTL epitope can be in vivo processed and correctly presented.

Evaluation of Immunogenicity and Protection Efficacy of the Recombinant Hypoxanthine-Guanine-Xanthine of Plasmodium falciparum in Mice / 中国寄生虫学与寄生虫病杂志

Objective To investigate immunogenicity and protection efficacy of the recombinant hypoxanthine-guanine-xanthine (HGXPRT) of Plasmodium falciparum expressed in Pichia pastoris. Methods 35 BALB/c mice were divided randomly into five groups: HGXPRT+ISA720 experiment group, HGXPRT+Freund experiment group, ISA720 adjuvant control group, Freund adjuvant control group, and blank control group. BALB/c mice were subcutaneously immunized three times with the HGXPRT protein formulated by either Freund or ISA720 adjuvants at a three weeks interval. Mice were bled via tail vein at 2 weeks after each immunization. Specific antibodies were detected by ELISA as well as IFAT using cultured parasites. The immunized mice were challenged with 105 P.yoelii 10 days after the third immunization and parasitemia was monitored daily by examining Giemsa-stained thin film. Results Strong immune response was induced by the HGXPRT antigen formulated with the adjuvant. Antibody titers of more than 1∶105 were detected after the third immunization while no specific antibody was detected in the mice immunized with adjuvants only. The antibodies against HGXPRT recognized the cultured parasite by IFAT. Four days after mice were challenged with P.yoelii, high parasitemia appeared in the two control groups, which were 24 h earlier than experiment groups. The mean parasitemia of HGXPRT+ISA720 experiment group(29.3%) was significantly lower than that of control groups (70.0%) (P

In Vitro Detection of Immune Sera to Plasmodium falciparum Chimeric Protein-2.9 for Inhibition of Parasite Growth by LDH Method / 中国寄生虫学与寄生虫病杂志

0.05). Conclusion The LDH method applied in the in vitro inhibition assay for P. falciparum seems simple, reliable and has generated a satisfactory result.

Malaria vaccine: current status and perspective / 第二军医大学学报

The emergency and rapid spread of drug resistant parasites and mosquitoes resistant to insecticides urgently demand new tools to control the disease. It is feasible to develop malaria vaccine based on animal and volunteers test. Scientists around the world are working on 3 types of malaria vaccine——pre erythrocytic, blood stage and transmission blocking. Some of the vaccine candidates are being tested in clinical trials and have been shown to be promising. It is very difficult to find a successful malaria vaccine due to its complex life cycle, antigen variations and multiple invasion pathways, but the vaccine will finally be developed.

Preparing domain Ⅱ F2 fragment of Plasmodium falciparum 175000 erythrocyte binding antigen and its combined immunization / 第二军医大学学报

Objective:To synthesize the eba 175Ⅱf2 gene of Plasmodium falciparum and express it in Pichia pastoris in the secreting form. The recombinant protein and PfCP 2.9 protein were used for combined immunization to see if there is antigen competition. Methods: Asymmetric PCR based metho d was utilized to synthesize the 959bp eba 175Ⅱf2 gene. Plasmid containing the synthetic gene was introduced into Pichi a pa storis by electroporation for inducible expression. The recombinant EBA 175Ⅱ F2 p rotein was purified by ion exchange and gel filtration ch romatography. Results: The eba 175Ⅱf2 gene was successfully ex p ressed in Pichia pastoris in the secreting form. The antibody titers in mice immunized with the combined EBA 175ⅡF2 and PfCP 2.9 proteins w ere much higher tha n those with individual protein by ELISA. Conclusion: No antigen competition is found when using the combined EBA 175ⅡF2 and PfCP 2.9 immunization in mice, indicating the potential of combining the 2 antigens for vaccination.

Constructing a pre-erythrocytic multi-epitope chimeric antigen of Plasmodium falciparum and its expression in Pichia pastoris / 第二军医大学学报

95%. Conclusi on : The synthetic PfCP TCL gene is expressed in Pichia pastoris and a method of easy purification is developed. The recombinant PfCP TCL protein provides a base for investigating its immune function and potential as a component of combined malaria vaccine.

Modification of inhibition assay for Plasmodium falciparum in vitro / 第二军医大学学报

0.05) were found between 2 method s. A modified in vitro inhibi tion assay without refreshing medium during a period of 72 h is established by r eduction of initial parasitemia from 1% to 0.2% 0.5%.

Expression of Plasmodium falciparum MSP1-31 fragment in attenuated Salmonella typhi CVD908 strain induced by tetracycline controlled promoter / 第二军医大学学报

Objective:To express MSP1-31 gene of Plasmodium falciparum in Salmonella typhi CVD908 vaccine strain using a tetracycline-controlled P LtetO promoter. Methods:The MSP1-31 gene was cloned into the plasmid of pZE11 and transformed into the CVD908/tetR strain by electroporation. Expression of MSP1-31 in CVD908/tetR strain was detected using the method of Western blot. Results: The recombinant plasmid of pZE11/MSP1-31 was constructed, there was effective expression of MSP1-31 protein in CVD908/tetR strain in presence of tetracycline, and no expression of gene in absence of tetracycline. Conclusion: The recombinant Salmonella typhi strain in which the expression of Plasmodium falciparum MSP1-31 fragment induced by tetracycline is established successfully.

Subcloning and Expression of Two Conserved Regions (Ⅰ, Ⅴ) on P190 Antigen of Plasmodium falciparum in E. coli / 第二军医大学学报

Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667) respectively, which encoded amino acid residues of conserved region I and V on the P190 antigen, were amplified by polymerase chain reaction from genomic DNA in FCC1/HN strain of Plasmodium falcipamm isolated from Hainan Province, China. It was found that there were five bases substitution in the P190CRV, in comparison with the nucleotide sequences of MAD20 strain. These two sequenced fragments were inserted into pGEX-2T plasmid. E.coli JM109 (DE3) were transformed with the recombinant plasmids and the parental plasmid. The results show that the two fragments were expressed at high level as C-terminal fusions with glutathione s-transferase (GST). The fusion proteins were easily purified from bacterial lysates by affinity chromatography using glutathione sepharose 4B.