[Protein interaction site of Toxoplasma gondii microneme protein 6 and aldolase determined by site-directed mutagenesis].

OBJECTIVE: To identify the protein interaction site of Toxoplasma gondii microneme protein 6 (MIC6) and aldolase by using site-directed mutagenesis. METHODS: Based on Toxoplasma gondii MIC6 gene sequence (GenBank Accession No. AF110270), the specific primers were designed. Tryptophan (W)-348 of MIC6 C terminus (MIC6C) was mutated to valine (V) via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE. RESULTS: The MIC6C W/N gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate. CONCLUSION: Tryptophan (W348) was the interaction site of MIC6 and aldolase in T. gondii.

Genotyping of Pneumocystis jirovecii isolates from Chinese HIV-infected patients based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes.

Genetic diversity of Pneumocystis jirovecii isolates based on internal transcribed spacer (ITS) of the nuclear rRNA locus has previously been reported. The information about ITS genotype and epidemiology of this organism in Chinese human immunodeficiency virus-infected patients has not been available. In this study, 12 bronchoalveolar lavage fluid specimens obtained from HIV-infected patients were analyzed by PCR followed by cloning, sequencing and typing. Three ITS1 genotypes (E, B and 'H') and four ITS2 genotypes (b, g, i and r) as previously reported were identified, the most common of which were E, b and i. Five ITS haplotypes (Eg, Eb, Bi, Er and 'H'r) and 19 new combination types were also identified with the most common types being Eg (four of 12 patients, 10 of 60 clones), Eb (three of 12 patients, 11 of 60 clones) and Bi (three of 12 patients, 10 of 60 clones). Nine patients were found to be co-infected with more than one ITS genotype of P. jirovecii. The prevalence of ITS genotypes in HIV patients from one Chinese hospital did not seem to be significantly different when compared to reports from other countries.

Molecular cloning and characterization of a matrix metalloproteinase, from Caenorhabditis elegans: employed to identify homologous protein from Angiostrongylus cantonensis.

Matrix metalloproteinases (MMPs) are a class of zinc-binding endopeptidases mainly responsible for degrading extracellular matrix constituent components, which also serve as effectors of leukocyte recruitment, cytotoxicity, cytokine and chemokine processing, and defensin activation involved in multiple mechanisms of immunomodulation. MMPs are a conserved proteolytic enzyme family participating in normal physiological function. In the present study, we have cloned a gene named CEMMP62 from Caenorhabditis elegans, the putative 62-kDa protein that contained 579 residues with MMP-conserved catalytic domain known as ZnMc_MMP and showed high identities with MMPs from other nematodes, and demonstrated that the recombinant CEMMP62 (rCEMMP62) expressed and purified from Escherichia coli could have weak proteolytic activity on swine gelatin; Western blot analysis revealed that sera from BALB/c mice immunized by recombinant protein could recognize excretory-secretary antigens from Angiostrongylus cantonensis third-stage larvae (L3). Also, the antiserum can recognize larval soluble antigens of L4 coming from mice (nonpermissive host) infected with A. cantonensis while it cannot recognize larval soluble antigens of L4 coming from rats (permissive host) infected with A. cantonensis. The results implied that probably CEMMP62 has homologous proteins which exist in A. cantonensis, and the potential MMP may play an important role in A. cantonensis infection and pathogenic process.

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay.

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.

[Protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down].

OBJECTIVE: To identify the protein-protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down. METHODS: The aldolase and actin genes were obtained from cDNA library by PCR amplification, and subcloned respectively into pGEX-4T-1 and pET30a. The fusion protein GST-Aldolase and Actin-His6 were expressed in E. coli upon induction by 1 mmol/L IPTG and then purified with affinity chromatography. Fifteen rats were immunized intradermally with 200 microg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 microg Actin-His6 protein per rat on the 2nd-4th immunizations. Rats were immunized for 4 times with 7 days interval. The serum of rats was collected from heart at the fifth day after the final immunization. Glutathione sepharose beads were incubated with GST-Aldolase protein, then incubated with Actin-His6, and bound proteins were eluted using sample buffer. Eluants were resolved by SDS-PAGE and Western blotting. RESULTS: The aldolase and actin genes were obtained, and the recombinant plasmid aldolase/pGEX-4T-1, actin/pET30a were successfully constructed. Protein GST-Aldolase and Actin-His6 were expressed and purified in vitro. Serum samples were prepared from rats immunized with protein Actin-His6, and polyclonal antibody was purified with affinity chromatography. SDS-PAGE and Western blotting analysis of products from GST pull-down experiment showed that the protein bands on NC membrane were specifically recognized by anti-Aldolase-His6 and anti-Actin-His6 antibody. CONCLUSION: Aldolase interacts with Actin of Toxoplasma gondii.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

[Epidemiological investigation of Angiostrongylus cantonensis in Jiangmen of Guangdong Province].

OBJECTIVE: To make an epidemiological survey on Angiostrongylus cantonensis in Jiangmen City of Guangdong Province. METHODS: From October 2006 to November 2007, the characteristics of A. cantonensis infection were investigated in Jiangmen district in various hosts, including the third stage larva infection in the snails Achatina fulica and Pomacea canaliculata by digestion method, and the adult A. cantonensis in rats by the dissection of heart and lungs. Relevant symptoms and dietary habits in Jiangmen residents who were randomly recruited were also investigated by questionnaire, and the specific IgG and IgM antibodies against A. cantonensis in their sera were detected by ELISA. RESULTS: 695 A. fulica and 720 P. canaliculata were examined. The infection rate of third stage larva of A. cantonensis were 45.0% and 1.8% respectively, with an infectivity of 53.74+/-147.30 and 5.23+/-8.51 respectively. Natural infection rate of A. cantonensis in all 229 rats was 4.4%. Among the 300 people surveyed, 11.3% had a history of eating raw or undercooked fish and shrimp, 5.3% directly or indirectly exposed to A. fulica or P. canaliculata. The positive rate of specific IgG antibody against A. cantonensis for serum samples among residents was 14.0% (42/300), and 5 serum samples in the 42 positive samples showed specific IgM antibody, with a positive rate of 1.7%. CONCLUSION: Jiangmen district is an endemic area of A. cantonensis, and the local residents are under the risk of infection.

[Pathological change in the brain of mice infected with Angiostrongylus cantonensis].

OBJECTIVE: To observe the pathological change in the brain of Angiostrongylus cantonensis-infected mouse. METHODS: Forty-eight mice were orally infected each with 40 third stage larvae of A. cantonensis, 3 mice were sacrificed at 7, 10, 13, 16, 19, 22, 25, 28 days postinfection respectively for worm recovery, and another 3 mice were for observing the histopathological change in tissue sections of the brain. RESULTS: Ten days postinfection, worms were found in the brain of the infected mice with a mean worm number of (7.0+/-1.7) per mouse. The highest number of worms was found at 16 days postinfection, with a mean of (23.7+/-4.9) per mouse. Notable symptoms of nervous system were seen on 15 days postinfection. Most mice died around 22 days postinfection. Histological examination revealed mechanical damages. Cavities and inflammation were observed in the brain parenchyma. Worms were seen in the subarachnoid space. Meningitis-like signs started at 13 days and aggravated then. CONCLUSIONS: Infection of A. cantonensis causes pathological change in mouse brain and the process is aggravating with postinfection time.

[Sequence analysis and expression of GRA7 gene of Toxoplasma gondii isolates in Escherichia coli].

OBJECTIVE: To analyze the difference of GRA7 gene of Toxoplasma gondii different isolated strains and express GRA7 in Escherichia coli. METHODS: The GRA7 gene was amplified from genomes of T. gondii isolates by PCR and was cloned into pGEX-4T-1. The recombinant plasmid was transformed into JM109 and sent to be sequenced. The sequence was analyzed with CLUSTALW (an internet tool). The recombinant plasmid was induced by IPTG to express the fusion protein,which was identified by SDS-PAGE and Western blot with positive sera. The protein was purified and used as a diagnostic antigen for ELISA to test serum samples. RESULTS: There was no difference among the sequences of T. gondii GRA7 gene from different isolates. The recombinant plasmid pGEX-4T-1/GRA7 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human and rabbit positive sera analyzed by Western blot. CONCLUSION: The GRA7 gene of T. gondii isolates is highly conservative. The GRA7 is expressed as a recombinant protein in Escherichia coli, which shows an immunoreactivity.

Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection.

BACKGROUND: A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj). METHODS: A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients. RESULTS: Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07 x 10(6) individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%. CONCLUSIONS: The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

[Study on low density lipoprotein receptor of adult Schistosoma japonicum].

OBJECTIVE: To provide a theoretical basis for the study of vaccines against Schistosoma japonicum, the receptor for human LDL in the tegument of adult Schistosoma japonicum was investigated. METHODS: Proteins existed in adult Schistosoma japonicum membrane were extracted by Triton X-100 and purified through reverse-phase high performance liquid chromatography (HPLC), and the main protein peaks were then collected separately. 125I-LDL of human plasma as the ligand, through the radioautography and radioligand binding assay, the protein which can bind human serum 125I-LDL specifically was identified. The molecular weight and IEF were detected by SDS-PAGE. RESULTS: According to the radioautography and radioligand binding assay, a protein with retention time of 10.5 min was proved to be able to bind human serum 125I-LDL specifically. SDS-PAGE revealed that the molecular weight of the purified protein is 60-65 kDa, and its IEF is 6.7. CONCLUSION: LDL binding protein may exist on the surface of both male and female adult Schistosoma japonicum with the function of obtaining cholesterol from host circulating system.

[Expression of proteinase cathepsin L1 gene of Schistosoma japonicum in Escherichia coli].

OBJECTIVE: To express the proteinase cathepsin L1 gene of Schistosoma japonicum (SjCL1) in Escherichia coli JM109 cells. METHODS: The SjCL1 gene was amplified from the recombinant plasmid pcDNA3-SjCL1 by PCR. The gene was cloned into a prokaryotic expression vector pGEX4T-1 to construct a recombinant plasmid pGEX-SjCL1. The E. coli JM109 cells were transformed with the recombinant plasmid pGEX-SjCL1 and the transformants were induced by IPTG to express the recombinant protein, the target protein was then identified by SDS-PAGE and Western blotting. RESULTS: A 1 kb length PCR product was obtained and a recombinant plasmid pGEX-SjCL1 was constructed. The expression product was detected by SDS-PAGE and Western blotting and an expression band about 62000 was found. CONCLUSION: The SjCL1 gene is effectively expressed in the E. coli JM109 cells.

[Screening and identification of phage antibodies from phage display library against Schistosoma japonicum].

OBJECTIVE: To obtain single chain variable fragment(ScFv) against the circulating antigen(CAg) from Schistosoma japonicum(Sj). METHODS: Metabolic antigen of adult worm of Sj (Sj-MAg) was used in the panning of phage library against Schistosoma japonicum. The activity of Sj-MAg-binding phage clones was assayed by ELISA. The specificity of expression products of the positive clones was analyzed by ELISA, SDS-PAGE and Western blotting. RESULTS: Seventy-two randomly selected clones were tested for the presence of anti-Sj-MAg ScFvs, 6 clones showed positive. The specificity of these 6 clones was confirmed by binding them to antigens of other four trematodes. Two clones (B04, C24) were found to bind to Sj-MAg but not to any of the antigens of other four trematodes and their expression products were about 31 kDa in size. CONCLUSION: ScFv antibodies against the circulating antigens from Schistosoma japonicum Sj-MAg can be selected and manufactured from the antibody library.

Absence of Pneumocystis jirovecii dihydropteroate synthase gene mutations among samples from a group of AIDS patients in China.

Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations are associated with failure of sulfur prophylaxis of Pneumocystis pneumonia. Here the P. jirovecii DHPS gene was amplified from bronchoalveolar fluid of 10 AIDS patients in China. No DHPS gene mutations were observed. The results suggest that the prevalence of DHPS mutations in China may be low.

[Construction of the life cycle of Angiostrongylus cantonensis in laboratory].

OBJECTIVE: To construct the life cycle of Angiostrongylus cantonensis (A.cantonensis) in laboratory condition. METHODS: SD rats were infected orally with the third-stage larvae of A.cantonensis collected from Jiangmen, Guangdong province. Six weeks after infection, the first-stage larvae were isolated from fresh feces of the rats by using Baermann funnel to infect 25 second-generation white jade snails raised in laboratory at the daily dose of 300 000 for 3 consecutive days. Three weeks later, the snails were dissected for counting the third-staged larvae of A.cantonensis, and those positive for A.cantonensis infection were fed directly to 10 fasting rats. The serum samples of the rats were then collected 2 weeks later for examination of specific antibodies using ELISA. The feces of the infected rats were examined microscopically after 6 weeks, and the brain, heart and lungs of the infected rats were dissected to observe the larvae at 3, 5, and 8 weeks, respectively. RESULTS: The 3-stage larvae of A.cantonensis were found in the second-generation snails 3 weeks after infection. The positivity rate of serum specific antibodies was 100% in the 10 rats 2 weeks after feeding of the infected snails. The 1-stage larvae were detected in the feces of the rats 6 weeks after infection, and the fourth-stage larvae were found in the brain of the rats at 3 weeks, while adult worm and eggs were found in the heart and lungs of the infected rats at 5 and 8 weeks. CONCLUSION: The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.

[Identification and tissue localization of intermediate filament protein in Angiostrongylus cantonensis].

OBJECTIVE: To identify the type of the intermediate filament (IF) protein of Angiostrongylus cantonensis and analyze its tissue localization. METHODS: Recombinant pET-IF of antigen IF was expressed in E.coli with IPTG induction, and the expression products were purified by His.Bind column and identified for determining the type of the IF protein by Western blotting. Anti-IF antibody was prepared by multi-spot subcutaneous injection into mouse and used to detect the tissue slices of A. cantonensis by immunohistochemical analysis. RESULTS: The antigen IF were correctly expressed and purified, and identified as a keratin located in the intestine wall and cytoplusma. CONCLUSION: The antigen IF is distributed in the intestine wall of A. cantonensis.