Search

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.

Objective: To prepare and purify polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and apply it to immunofluorescence localization., Methods: The constructed recombinant plasmid pET32a-ROP2 was transformed into E. coli BL21 (DE3) and the protein was expressed under the condition of 0.5 mmol/L IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body, respectively. The washed inclusion bodies were dissolved in urea. New Zealand White rabbits were immunized with 200 microg purified recombinant ROP2 mixing with the same volume of Freund's adjuvant for 3 times at interval of 14 days and 19 days, respectively. Rabbit serum was collected at 10 days after the last immunization. Polyclonal antibody in rabbit serum was purified with HiTrap Protein G HP affinity purification column. Indirect ELISA and Western blotting were used to detect antibody titer and specificity of polyclonal antibody against the recombinant ROP2. The polyclonal antibody was used to the localization of ROP2 on the parasitophorous vacuole membrane in human foreskin fibroblasts infected by Toxoplasma tachyzoites by the immunofluorescence method., Results: The recombinant ROP2 protein was obtained and specific rabbit-derived polyclonal antibody was prepared. Indirect ELISA confirmed that the rabbit-derived polyclonal antibody titer reached 1:102400, and the recombinant ROP2 protein was recognized by specific polyclonal antibody. Immunofluorescence localization test showed that the ROP2 protein was located on the parasitophorous vacuole membrane., Conclusion: The rabbit-derived polyclonal antibody against ROP2 is prepared, and used in immunofluorescence localization of ROP2 on parasitophorous vacuole membrane.

Objective: To separate and identify the surface proteins and secreted proteins of Toxoplasma gondii tachyzoites of RH strain., Methods: T. gondii tachyzoites were cultured in Vero cells, and purified by filtration and Percoll cell separation solution. The biotin-labeled tachyzoites were lysed, and the surface and secreted proteins were separated by NeutrAvidin agarose beads. After condensation and SDS-PAGE, the protein were collected, digested and identified by LC/MS-MS., Results: A total of 785 T. gondii proteins were identified, 81 (10.3%) of which were originally annotated as the surface or secreted proteins. Among the highly-expressed (PSM>10) 65 proteins, 43 (66%) were originally annotated as surface or secreted proteins, while the others were predicted unknown proteins., Conclusion: The surface and secreted proteins of T. gondii are separated by biotin labeling and avidin chromatography, among which some potential new surface or secreted proteins of T. gondii are identified.

Objective: P35 is an important surface protein for Toxoplasma gondii. To obtain the highly pure and specific antigenicity protein, the gene P35 was cloned and its product was expressed in E. coli Rosetta. The expressed protein was purified and its immunogenecity was studied., Methods: A pair of primers was designed according to cDNA sequence of P35, and then the P35 gene amplified by PCR was cloned into the vector pGEM-T and proved by DNA sequencing. The P35 gene was subcloned into prokaryotic expression vector pET-KDO, its expression was induced by IPTG, and the target protein was obtained by affinity chromatography., Results: The P35 gene was successfully amplified from genome DNA of Toxoplasma gondii RH strain and a fusion protein was expressed in E. coli. The molecular weight of the expressed protein was about Mr 42 000. Western blotting indicated that the antigenicity of the protein was specific., Conclusions: The plasmid pET-KDO-p35 is constructed and the high efficient expression of P35 fusion protein has been achieved in E. coli. The fusion protein shows a specific antigenicity, the P35 fusion protein has a potential value in the diagnosis of toxoplasmosis.

Objective: In order to find the best extraction method for proteins of Alexandrium tamarense for two-dimensional electrophoresis (2-DE) analysis., Methods: Three methods for extracting proteins from A. tamarense were compared, including trihydroxymethyl aminomethane (Tris-HCl ) buffer extraction, trichloroacetic acid (TCA)/acetone precipitation and lysis buffer extraction. Alga was cultivated in normal f/2 media (control) and supplemented with algicidal substances. Proteins obtained using the best extraction method were separated with 2-DE. Protein-expression differences were identified using matrix-assisted laser desorption/ionization-time of flight ( MALDI-TOF) mass spectrometry (MS)., Results: Among the three protein extraction methods, lysis buffer extraction shows the best detection of the number and quality of protein spots with a clear background. Then, the lysis buffer extraction method was successfully applied to profiling protein expression in algicidal substances stress conditions and 14 differential expression proteins were identified using MALDI-TOF/MS., Conclusion: Lysis buffer extraction was the most effective protein extraction method for Alexandrium tamarense.

Objective: To clone and express BC48 gene of Babesia caballi, and to establish an indirect ELISA for the diagnosis of B. caballi in equine animals., Method: The genomic DNA of B. caballi was extracted from the infected donkey blood. BC48 gene was amplified by PCR. The PCR product was cloned into expression plasmid pET28a, and expressed in E. coli BL21 with IPTG induction. The recombinant protein was purified by Ni-NTA affinity chro-matography and was used as a diagnostic antigen to establish an indirect ELISA. The reaction conditions of the indirect ELISA were optimized. Specificity and sensitivity of this method were evaluated., Result: BC48 gene of B. caballi was 1 272 bp. The recombinant protein was expressed in E. coli BL21 as a soluble protein with a molecular weight of about M, 46 000 under induction of IPTG. The concentration of purified protein was 12.98 mg/ml. The best conditions were obtained for the ELISA when the antigen concentration was 65 microg/ml with the serum dilution of 1:80. The protein specifically reacted with serum from donkey infected by B. caballi, but did not react with serum from donkey infected by Theileria equi (B. equi). Both ELISA and microscopy were applied to examine 17 donkeys in the field, 3 were positive by ELISA and 2 were found parasite-positive, respectively., Conclusion: The indirect ELISA method may be used to detect B. caballi infection in equine animals.

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.

Objective: To amplify ROP2 from the genomic DNA of Toxoplasma gondii RH strain and construct eukaryotic expression plasmid pc-DNA3-ROP2., Methods: Tachyzoites of T. gondii RH strain were collected and depurated to obtain genome. A pair of primers was designed and synthesized according ROP2 gene sequence. The gene fragment encoding ROP2 was amplified from the genomic DNA of T. gondii RH strain by means of PCR. It was then reclaimed and purified, and inserted into cloning vector pUCm-T. The recon was cut by EcoR I, Hind III, and the inserted ROP2 gene fragment was subcloned into pc-DNA3 eukaryotic expression vector using T4DNA ligase, followed by further PCR identification, double digestion via restrictive enzymes, and sequencing., Results: The amplified specific gene fragment of ROP2 was about 1.7 kb in length. The gene fragment cloned and subcloned into pc-DNA3 was correct, and the eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed., Conclusion: The recombinant plasmid pc-DNA3-ROP2 was successfully constructed.

Objective: To construct a prokaryotic expression system containing the dense granule protein 4 (GRA4) of Toxoplasma gondii, purify the expressed protein and detect its immunogenicity., Methods: The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His Bind affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA., Results: The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40,000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T. gondii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody., Conclusion: The pET-GRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.

Objective: To clone and express a homologue of human macrophage migration inhibitory factor (MIF) from P. falciparum 3D7--PfMIF., Methods: The nucleotide sequence of PfMIF was found through blast P. falciparum genomic sequence databases with the amino acid sequence of human MIF (HuMIF). RT-PCR, DNA sequencing, and bioinformatics analysis were used for the cloning of Pfmif gene. The recombinant protein was expressed in E. coli and purified through the affinity column., Results: The full length of Pfmif gene was cloned and sequenced. It was composed of 351 nucleotides and encoded 116 amino acids with the typical characteristic of MIF family. The recombinant protein was successfully expressed and purified., Conclusions: The Pfmif gene and recombinant protein were successfully isolated and PfMIF was preliminarily identified as a novel member of MIF family.

Objective: To express truncated SAG1 gene in Escherichia coli to obtain purified recombinant SAG1, and identify the immunoreactivity of the product., Methods: The plasmid pET-30a(+)-trSAG1 was constructed, which was cut by Nco I and HindIII to obtain truncated SAG1 gene and inserted into pET-32a(+) cut by the same two restriction enzymes. After identification by restriction enzymes, the plasmid pET-32a(+)-trSAG1 was transformed into E.coli BL21, and the soluble product induced by 0.1 mmol/L IPTG for 4 hour before purification with Ni-NTA agarose, followed by identification of the purified recombinant protein with SDS-PAGE, Western-blotting and ELISA., Results: Recombinant pET-32a(+)-trSAG1 was successfully constructed and high level of truncated SAG1 expression achieved in E.coli. SDS-PAGE showed that the expressed protein was approximately 40,000 in size and expressed in a soluble form that could be easily purified by Ni-NTA agarose. Western-blotting demonstrated that the purified product could be specifically recognized by sera from rabbits immunized with native antigen of T.gondii, and could also be recognized, as shown by ELISA, by sera from human with T.gondii infection., Conclusions: The truncated SAG1 gene was successfully expressed in E.coli in a soluble form, and the recombinant protein may be of value in the diagnosis of toxoplasmosis.

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

Aim: To isolate and quantitate chloroquine (CQ)-binding proteins in both CQ-sensitive (CS) AND CQ-resistant (CR) Plasmodium berghei., Methods: P. berghei infected mice were each given i.g. CQ 400 mg/kg, respectively, 3 h later, the proteins of the parasites collected were separated using Ultrogel AcA34 gel column, followed by CQ extraction, the CQ concentration was determined by HPLC (extra standard)., Results: There were 58 protein peaks in the CS, all of which were bound with CQ, fraction Nos. 64-86 (peak 9) being the predominant, accounting for 16.4% of the total amount of CQ-binding protein. In the CR there were 40 protein peaks, among which 32 were bound with CQ, the predominant fraction Nos. 60-83 (peaks 6, 7), had a higher CQ-binding ability than CS., Conclusion: The distribution and amount of each CQ-binding protein peak in both CS and CR strains of P. berghei were demonstrated, exhibiting differences in the ability of CQ-binding not only between CS and CR strains but also within the same strain.

Aim: Determining nucleotide sequence of the circumsporozoite protein partial gene of the Plasmodium falciparum PFD-3/YN (Yunnan of China) and finding out the differences of the CS gene sequence between Chinese Plasmodium falciparum isolate and other isolates., Methods: The circumsporozoite protein gene fragment was amplified by polymerase chain reaction and cloned into M13 bacteriophage. M13-CSP single strand DNAs of the three positive clones were extracted respectively. Then, the nucleotide sequence of the CS gene fragment was determined by the dideoxy chain termination method. PCGENE software was used to compare and analyze the CS gene sequence of the six isolates., Results: Different degrees of diversity of the CS gene sequences were found among P. falciparum PFD-3/YN and other isolates(T4, Wellcome, NF54, 3D7 and 7G8). A non-silent substitution at the nucleotide level being found in the P. f Th/Tc antigenic epitope region., Conclusion: There were differences in the CS gene sequence among P. falciparum PFD-3/YN and those of other isolates.

Aim: To develop a genotyping method based on amplifying glutamate-rich protein (GLURP) gene for the diagnosis and identification of Plasmodium falciparum., Methods: Two pairs of primers specific for GLURP gene of P. falciparum were designed and synthesized. R2 polymorphic domain of GLURP gene was amplified by nested PCR, which was applied to genotyping of P. falciparum isolates obtained from patients attending the malaria clinic at the village of Borai, Thailand., Results: Conspicuous polymorphism of GLURP alleles in natural populations of P. falciparum was found. 290 GLURP alleles were detected in 154 P. falciparum infections. Among the above-mentioned alleles, 12 different GLURP genotypes were distinguished according to different DNA sizes. Of them, the most frequently found allele was a variant of 770 bp, the least allele was that of 1,100 bp. More than 43% of the patients were found to be infected with mixed alleles. No apparent change for frequencies of the 12 different alleles was found in the 9-month longitudinal study., Conclusion: A genotyping method is developed for the research of strain taxonomy and pathogenesis of malaria parasites.