Characterization of a Clonorchis sinensis antigen, calmodulin, and its relationship with liver fibrosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.</p><p><b>METHODS</b>The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.</p><p><b>RESULTS</b>The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.</p><p><b>CONCLUSION</b>The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.</p>

Sequence analysis,cloning expression and immunogenicity analysis of lactate dehydrogenase gene from Taenia solium / 中国人兽共患病学报

The structure and properties about encoding protein of lactate dehydrogenase A from Taenia solium(Ts LDH-A)were analyzed and predicted by bioinformatics in this study.The immunological characteristics of this novel gene were also analyzed by cloning and expressing.The full-length cDNA encoding Ts LDH-A was identified from the cDNA plasmid library by blastx and rpsblast programs provided by NCBI.The physico-chemical properties and structures of Ts LDH-A were analyzed by tools provided by ExPASy.And the B cell epitopes of Ts LDH-A were predicted by the B Cell Epitope Prediction Tools provided by IEDB Analysis Resource.The PCR amplified coding region of Ts LDH-A was cloned into the prokaryotic expression vector pET-28a (+) and expressed in E.coli BL21 with IPTG induction.The immunogenicity of the purified recombinant protein was analyzed by Western Blotting.It was demonstrated that the amino acid sequence of Ts LDH-A had identity with that of LDH-A from other specie and there was a conserved LDH domain in the deduced amino acid sequence.The full-length cDNA sequence encoding Ts LDH-A included a complete open reading frame(ORF)of 1332 bp and coded to a putative protein with 331 amino acids.The molecular weight of Ts LDH-A was predicted to be 35461.1 Da and the coding protein was demonstrated to contain 3 trans-membrane regions and 4 main B cell epitopes.The active site of L-lactate dehydrogenase located at the epitope aa190-199.The 3 key residues in the catalytic site of enzyme were conserved in different species and located near to each other in spatial position.PCR,double enzyme restriction and DNA sequencing were used to identify pET28a (+)-Ts LDH-A.The recombinant protein could react with the rat's sera as well as the sera from the patients and the swine infected Taenia solium.It is clear that the full-length cDNA sequence encoding Ts LDH-A can be screened from the cDNA library of adult Taenia solium by bioinformatics analysis and can be used to investigate the structure and properties about gene and encoding protein of Ts LDH-A as well as the immunological activities of gene expression in the prokaryotic system.

Bioinformatic analysis on the Rap2B-like gene from Clonorchis sinnensis and its cloning and expression / 中国人兽共患病学报

To analyze the structural characteristics of the Rap2B gene from Clonorchis sinensis by bioinformatics and to clone and express this gene through genetic engineering methods in order to obtain the corresponding recombinant protein.The structural and functional characteristics of the Rap2B protein were analyzed by using the related bioinformatic softwares. Using the full-length cDNA plasmid clone (Noc009e03) as template , the coding region of Rap2B gene sequence was amplified with PCR and cloned into the prokaryotic expression vector pET-28a(+) and then expressed in E.coli BL21 through IPIG induction. The recombinant protein expressed was purified by Ni-IDA affinity chromatography and detected by SDS-PAGE and Western blotting. It was demonstrated that the size of this gene was 847 bp in length, encoding 141 amino acids and its theoretical molecular weight was 15851.1 daltons. As demonstrated by PCR, double enzyme digestion and DNA sequencing, the recombinant expression plasmid pET-28a(+)-Rap2B was successively constructed and the Rap2B-like gene was highly expressed in E.coli BL21. The recombinant protein was obtained through purification with His affinity chromatography and could react specifically with the serum of rats infected with C.sinensis. Through these investigations, the Rap2B protein may be predicted as a member of the Ras oncogene family protein and is potential in the carcinogenic process of C.sinensis.

Establishment and characteristics of hybrid embryonic stem cell lines from blastocysts of the (C57BL/6J × 129/J)F1 mouse / 中国病理生理杂志

AIM: To establish hybrid mouse embryonic stem (ES) cell line from blastocysts of the (C57BL/6J × 129/J) F1 mouse. METHODS: 3.5 days post- coitus (d.p.c.) blastocysts were cultured on mouse embryonic fibroblasts (MEFs) in the medium, after 3 - 4 days, Inner cell mass were picked up and disaggregated, then reseeded. After the ES - like colonies appeared, passaged them to give permanent ES cell lines. The pluripotent propertes of ES cells obtained were analyzed by alkaline phosphatase (AKP) activity, expression of SSEA- 1 and Oct-4, and their capacity to form teratoma. RESULTS: Two hybrid ES cell lines, SC1001, SC1002 were obtained from blastocysts of the (C57BL/6J × 129/J) F1 genotype. Most of these ES cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines were analyzed on the basis of their alkaline phosphatase activity, expression of SSEA - 1 and Oct - 4, and their capacity to form teratoma in severe combined immunodeficiency (SCID) mice. CONCLUSION: Two hybrid mouse ES cell lines having pluripotent properties and capacity for long - term self renewal were generated from blastocysts of the ( C57BL/6J × 129/J) F1 genotype.

Cloning, expression and antibacterial activity of histone H1 and its N-terminal peptide from Carassius auratus / 生物医学工程学杂志

In order to determine whether H1 histone proteins are associated with innate immune antimicrobial response in goldfish, we extracted the total RNA from the hemocytes of goldfish (Carassius auratus), designed 3 pairs of primers based on the previous antimicrobial peptide sequences from fish and performed RT-PCR. Among 3 obtained-PCR products, we identified a novel histone H1 coding sequence of 576 bp which belongs to the histone H1 family and is 78% homologous with the amino acid sequence of histone H1 from Salmon salar that had been found with an important role in salmon defenses against infectious pathogens. The H1 histone of goldfish contained 3 predicting cleavage sites that divided the protein into 4 parts. We successfully cloned and expressed the whole CDs (No ATG) of H1 histone and its N-terminal part (2-38aa) in Pichia pPIC9K expression system. The products of H1 histone and its N-terminal deriving peptide (AEVAPAASAPPAKAPKKKSAAKAKKAGPAVGDLIVKA) show antimicrobial activity. The results suggested that the H1 histone fragment reported in this paper is a novel antimicrobial peptide found in goldfish. H1 histone plays an important role in innate immune responses of goldfish.

Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases.</p><p><b>METHODS</b>Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells.</p><p><b>RESULTS</b>Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect.</p><p><b>CONCLUSION</b>siRNA may be effective in inhibiting SARS-associated coronavirus replication.</p>

Primary Study of the Structure and Antigenic Epitopes of Antigen Pf332 / 热带医学杂志

Objective To understand the primary structure and potential antigenic epitopes of antigen Pf332(Ag332) of P.falciparum iso late FCC1/HN.Methods　Based on the published Pf332 gene sequence , nine pairs of primers were designed for the PCR amplification of the Pf332 gen e fragments from genomic DNA of P.falciparum isolate FCC1/HN. The amplified gene fragments were subcloned into pMD-18T vectors and sequenced. The sequences were aligned using DNAstar software to obtain the full-length sequence of the gene Pf332. The primary structure and sequence homology of Ag332 were analyzed by SAPS, Tmpred, SingalP and Blastn programs. Three fragments, R0, R1 and R2, cor responding to nt#9595-10083, nt#10339-10767 and nt#10855-11247 of Pf332 gene were subcloned into the eukaryotic expression vector pcDNA3-S separately. The Balb/c mice were immunized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3- S-R2 separately, and the expressions of the recombinant proteins were detected by immunohistochemistry assay. The protective immune responses elicited by DNA I mmunization were analyzed by ELISA and parasite growth inhibition tests in vitro .Results　Nine Pf332 gene fragments were specifically amplif ied, subcloned into pMD-18T vectors and sequenced. Pf332 gene of the P.falci parum isolate FCC1/HN was 16,377 bp in length, encoding a protein of 5,458 ami no acids, about 615.28kDa. The Ag332 contains 17 regions of highly degenerated Glu-rich repeats, with 30.18% Glu in total amino acids of Ag332. Ag332 of P.falciparum isolate FCC1/HN and 3D7 exhibited 94.55 % homology in amino acid residues. The results of immunohischemistry assay showed that R0, R1 and R2 were expressed in mice muscle tissue. The amount of IgG antibody of the groups immu nized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 were higher than those of blank and pcDNA3 groups (P＜0.05). The result of parasite growth inhibition test showed that the immunized sera at 1∶5 dilution of groups of pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 had an incomplete inhibitor y effect on P.falciparum growth. Conclusion　The antigen Pf332 is an large protein containing highly degenerated Glu-rich repeats. Pf332 gene fragments, R1 and R2 encoding potent antigenic epitope repeats.

Generation and analysis of 113 adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To rapidly and economically obtain knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes using expressed sequence tag (EST).</p><p><b>METHODS</b>A directional cDNA library constructed from Schistosoma japonicum (Chinese strain) adult stage RNA was used to generate expressed sequence tags (ESTs). These were compared against an EMBL-parasites database and GENBANK database by BLASTn and BLASTx.</p><p><b>RESULTS</b>A total of 314 phage clones were randomly selected for generating expressed sequence tags (ESTs). From these clones, 132 EST-quality sequence were obtained. Among these EST-quality sequences, 113 ESTs were successfully submitted to the dbEST at GenBanK. A total of 7.6% of these EST-quality sequences were previously identified sequence of Schistosoma japonicum, while 4.5% were putatively identified sequences of Schistosoma japonicum. A total of 23.5% of these EST-quality sequences were putatively identified sequence of Schistosoma mansoni or other organisms. 57.6% had no matches in the database and were classified as unknown sequences. Most ESTs with the putative protein identified belonged to housekeeping proteins. Information about several interesting genes was found.</p><p><b>CONCLUSION</b>Partial cDNA sequencing to generate expressed sequence tags (ESTs) has the potential to rapidly and economically increase our knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes.</p>

Construction of Eukaryotic Expression Recombinant Plasmid and Sequence Analysis of p41-3 Gene of Plasmodium Falciparum Isolate FCC1/HN / 中山大学学报(医学科学版)

[Objective] To construct a eukaryotic expression plasmid containing a gene encoding a 41-3 kilodalton blood stage antigen (p41-3) of Plasmodiu falciparum isolate FCC1/HN, and to determine the sequence of p41-3 gene and analyze the homology of the sequences of 341-3 gene of different P. falciparum isolates. [ Methods] Two pairs of primers were designed according to the known sequence of p41-3 gene. Using PCR technique, the p41-3 gene was obtained by amplification from genomic DNA of isolate FCC1/HN. By cloning target gene into a eukaryotic expression vector, pcDNA3, a recombinant plasmid pcDNA3-p41-3 was con structed and trarsferred into E. coli DH5α. The positive clones were screened and identified by agarose gel electrophoresis, endonu clease digestion and PCR technique. The correct recombinant plasmid pcDNA3-p41-3 was used as template, and the nucleotide se quence of p41-3 gene was determined by the dideoxy chain termination method. Using softwares to analyze the structure and sequence homology of p41-3 gene between isolate FCC1/HN and FCBR. [Results] The p41-3 gene was specifically amplified from genomic DNA of Plasmodiumm falciparum isolate FCC1/HN, and the correct recombinant plasmid pcDNA3-p41-3 was screened and identi fied. The result of sequence determination showed that the p41-3 gene of isolate FCC1/HN was 2 137 base pairs in full length, encod ing 375 amino acids. Isolate FCC1/HN and isolate FCBR exhibited 98.98 % homology in the nucleiotide sequences and 99.73 % ho mology in the encoded anino acids of p41-3 gene. [Conclusion] The eukaryotic expression plasmid pcDNA3-p41-3 is successfully con structed and nucleotide sequence of p41-3 gene of isolate FCC1/HN is determined. The p41-3 genes of isolate FCC1/HN and isolate FCBR share quite high homology.

Detection of CTP gene expression in the asexual erythrocytic stages of Plasmodium falciparum (FCC1 /HN) by reverse transcriptase- polymerase chain reaction and construction of eukaryotic expression vector of CTP gene / 热带医学杂志

Objectve To detect whether the CTP(phosphocholine cytidylyltransferase) gene was expressed in the asexual erythrocytic stages of Plasmodium falciparum (FCC 1/HN )by using the RT - PCR and to construct eukaryotic expression vector of CTP. Method The erythrocytic stage parasites of Plasmodium falciparum were cultured as described by Trager and Jensen. RNA from erythrocytic stage parasite was extracted by using Trizol reagent. The complete genes coding for CTP gene isolates FCCI/HN were amplified by reverse transcriptase -polymerase chain reaction(RT- PCR). CTP gene was cloned into eukaryotic expression vector pcDNA3. Results CTP encoding gene was amplified from the erythrocytic stages of Plasmodiumfalciparum (FCC 1/HN) and eukaryotic expression vector of CTP was constructed. Conclusion CTP gene was expressed in the erythrocytic stages of Plasmodium falciparum (FCC 1/HN) and eukaryotic expression vector of CTP was successfully constructed.

Obtaining, analysis and cloning of a novel gene of Schistosoma japonicum / 中华传染病杂志

Objective To obtain and characterize a novel gene of Schistosoma japonicum. Methods Looking for a novel gene through expressed sequence tags(EST),then predicting its function with the help of software on line. Finally the novel gene was cloned into an eukaryotic plasmid pEGFP N3. Results A novel gene with complete ORF was obtained. It has homogeneity with Defender Against Apoptotic Death 1 of human and pig. Conclusions EST is a good method to find new genes and analyse them with the help of software on line and characterize their function.

IMMUNE RESPONSES OF MICE VACCINATED WITH RECOMBINANTS CODED DIFFERENT STAGE GENES FROM FCC 1/HN STRAIN OF PLASMODIUM FALCIPARUM / 中国人兽共患病学报

Aim To search the effective malaria mixed gene vaccine and to explore the immune response of vaccinated host. The recombinant pcDNA3-EBA175/HRP Ⅱ was injected alone or mixed with pcDNA3-Pfs25 of Plasmodium falciparum into mice by intramuscular route. The muscle of injected parts were treated previously, e.g. injecting 50μl of 0. 5％bupivacaine into the muscle of left latter leg, with 2mm depth. To observed the changes of IgG antibody value, the splenic lymphocyte proliferation, the ratio of CD4+/CD8+ subgroups and NK cell killing activity. After injecting recombinant pcDNA3-EBA175/HRP Ⅱ alone or mixed with pcDNA3-Pfs25 into mice by intramuscular route, it showed that sera IgG value increased, the splenic T lymphocyte proliferation stimulated by specific antigen of Plasmodium falciparum increased, the ratio of CD4+/CD8+ decreased and NK cell activity increased. Enhanced immune injection could improve host's immune reaction.It is suggested intramuscular injection is an effective immune route, and mice inoculated with coding two gene recombinant alone or that mixed with sexual stage gene recombinant could all induce increased humoral and cellular immune response, and NK cell activity.

T CELL IMMUNE RESPONSE TO THE CIRCUMSPOROZOITE PROTEINOF PLASMODIUM FALCIPARUM, SOUTHERN CHINA ISOLATE FCC/HN AND ANTIGEN-INDUCED HL-2 AND IFN-γ Production ANALYSIS OF SPECIFIC / 中国人兽共患病学报

Inthis study, the DNA fragment encoding the regions Ⅰ to Ⅱ of CSP gene from Plasmodium falciparm isolate FCC1/HN was cloned into an. expression vector pcDNA3 contained cytomegalovius (CMV) and transformed into human Hela cell line. The expressed protein PfCSP (condidate vaccine) was used to immunize BALB/c mice by subcutaneous、 intravenous or intraperitoneal administration respectively. The splenocyte of BALB/c mice immumized with the condidate vaccine released significantly IL-2 and IFN-γ following stimulation with this vaccine. It is associated with increase of the splenic T lymphocyte proliferation stimulated by this vaccine and enhancement of NK cell killing activity in the former studies. These results suggested that the vaccine could stimulate T cell response and enhance the cell-mediated immunity.

SEQUENCING AND HOMOLOGOUS ANALYSIS OF THE SEXUAL STAGE PFS25 GENE FROM PLASMODIUM FAL CIPARUM PFD-3/YN ISOLATE / 中国人兽共患病学报

Malaria vaccines are being developed against different stages in the parasite's life cycle. Although not directly protective ,the sexual stage vaccines would induce antibodies that would prevent infection of mosquitoes when ingestedin a bloodmeal containing sexual stage parasites. pfs25 has been tested to be an important candidate antigen for malarial transmission blocking vaccine . In this report we analyzed the complete code of pfs25 gene of Plasmodium falciparum PFD-3/YN isolate. The result shows that the gene encoding pfs25 of PED-3/YN isolate has a mutant which generates a PstI endonuclease restriction site and shares 99.2％ nucleotide homology with that of NF4 isolate.

Cloning, sequence analysis and expressing of LytA gene from different strains of Streptococcus pneumoniae / 中华传染病杂志

Objective To clone the gene of autolysin(LytA) which are from different clinical strains of Streptococcus pneumoniae and express them in Escherichia coli. Methods The LytA gene was amplified by PCR from the total DNA of S.pneumoniae. Primers were designed according to the LytA gene sequence of R6. Recombinant plasmids were constructed and the sequences of different clinical strains were analyzed through method of bioinformatics. The cloned genes were expressed in E.coli and detected by SDS-PAGE. Results Complete LytA gene were amplified from all of the different clinical strains of S.pneumoniae and recombinant plasmids pGEX-4T-1-LytA were constructed successfully. After comparing the sequence of DNA and supposed protein, we find some differences. Induced by IPTG, LytA gene was expressed effectively in E.coli Jm109. Result of SDS-PAGE showed that the molecular weight of expressed protein was 62 kD, the same as calculated. Conclusions The sequences encoding LytA from different clinical strains of S.pneumoniae were cloned, the recombinant plasmids pGEX-4T-1-LytA was constructed successfully. Sequence analysis showed that there have difference among the gene and amino acid sequences of LytA from different clinical strains. Further studies should be focused on whether the difference contributes to activity of autolysin and the drug-resistance of S.pneumoniae.

Parasite-origin IgE-dependent Histamine-releasing Factors in Inducing Histamine Release from Sensitized Mast Cells / 中国寄生虫学与寄生虫病杂志

Objective To obtain the recombinant IgE-dependent histamine-releasing factors of Schistosoma japonicum and Clonorchis sinensis (rSjHRF and rCsHRF) and to study the effect of recombinant HRFs to induce histamine release from sensitized rat mast cells. Methods The complete coding regions of SjHRF and CsHRF were cloned separately, and the recombinant plasmids were respectively transformed and expressed in BL21 cells. The soluble recom-binant rSjHRF and rCsHRF were purified. Aliquots of the mast cells obtained from the lungs of OVA-immunized rats were separately incubated with rSjHRF and rCsHRF and the released histamine was measured by the OPT spectrofluorometric procedure. The dose-dependent curves and the kinetics of histamine release induced by rSjHRF and rCsHRF were prepared. Results The recombinant plasmids pET-30-rSjHRF and pET-30-rCsHRF were constructed successfully and the purified soluble recombinant proteins rSjHRF and rCsHRF were obtained by affinity chromatography. rSjHRF and rCsHRF induced histamine release from sensitized mast cells in a dose-dependent manner. At the concentration of 150 mg/L, the average rate of histamine release from sensitized mast cells induced by rSjHRF and rCsHRF were 49.78% and 32.63%, respectively. Histamine release increased with prolonged reaction time and the maximal release occurred at 35min. Conclusion The recombinant parasite-originated IgE-dependent HRFs show an effect of inducing histamine release from sensitized mast cells, suggesting that this protein would play a role in type I hypersensitivity in hosts with parasitic infections.

CLONING AND SEQUENCE ANALYSIS OF RESA GENE FRAGMENT OF PLASMODIUM FALCIPARUM ISOLATE FCC1/HN / 中国寄生虫学与寄生虫病杂志

Objective] To determine the nucleotide sequence of the 3′\|termal of the RESA gene Plasmodium falciparum isolate FCC1/HN, and find out the differences of the sequences of RESA gene among isolate FCC1/HN,FC27,NF7 and Palo Alto. [Methods] 3′\|terminal fragment of RESA gene of P.falciparum isolate FCC1/HN was amplified by PCR method, then was cloned into pMD18\|T vector. The recombinant was screened and identified by BamHI+XhoI and PCR technique. The nucleotide sequence of the 3′\|terminal of the RESA gene was determined by the dideoxy chain termination method. DNASTAR and BLAST software were used to compare and analyze the RESA gene sequences among the different isolates. [Results] The 3′\|termal fragment of the RESA gene with about 846 bp was specifically amplified by PCR, the recombinant pMD18\|T\|RESA was successfully constructed. Different degrees of diversity of the RESA gene sequences were found among P.falciparum isolates FCC1/HN、FC27,NF7 and Palo Alto. [Conclusion] There were differences in the sequences of RESA gene among the P.falciparum isolate FCC1/HN and three other isolates (FC27,NF7 and Palto alto).

AMPLIFICATION, CLONING AND EXPRESSION OF A GENE ENCODING HEXOSE TRANSPORTER OF PLASMODIUM FALCIPARUM / 中国寄生虫学与寄生虫病杂志

Objective] To amplify,clone and express of a gene encoding hexose transporter of Plasmodium falcipuram(PfHT1) from Southern China isolate FCC1/HN for studing the immune of recombinant which protective from malaria parasite infection. [Methods] Cultivation of P.falciparum isolate FCC1/HN in vitro; extraction of genomic DNA from FCC1/HN using the alkali specific cleavage method; PCR amplification of PfHT1 and cloning into eukaryotic expression vector, pEGFPN3. The recombinant was introduced into mammalian cells, HEPG2 by using liposome\|mediated transfection. [Results] The gene encoding PfHT1 was specifically amplified from the genomic DNA of P.falciparum isolate FCC1/HN. The size of amplified fragment was 1 516 base pair. The eukaryotic expression recombinant, pN3\|HT1 , was constructed and expressed steadily in the hepatocarcinoma cell lines, HEPG2. [Conclusion] The gene encoding PfHT1 was successfully amplified and cloned. The pN3\|HT1/HEPG2 cell line was built for expressing fusion protein of GFP\|HT1.

Amplification of SAG3 gene fragment and its application in detection of toxoplasma gondii in hemato- poietic stem cell transplantation / 中华器官移植杂志

Objective To establish a rapid, specific and sensitive diagnostic technique for the human T. gondii infection with hematopoietic stem cell transplantation and discuss its clinical significance. Me- thods Fifty-six patients subject to hematopoietic stem cell transplantation were detected with ELISA and PCR. Results Among 56 recipients of hematopoietic stem cell transplantation, 7 were positive for T. gondii antigen and 10 were positive for SAG3 gene fragment respectively with the positive rate being 14.3 % and 17.8 % in the ELISA and PCR screening respectively. Twenty healthy people were negative for anti-Toxo antibody.Conclusion PCR is an accurate, relatively rapid, sensitive and specific method for detecting SAG3 gene of T. gondii, and can be considered a valuable additional tool for identification of T. gondii infections.

Identification of the SjARG Novel Gene and Study on Its Protective Potential as a Vaccine / 中国寄生虫学与寄生虫病杂志

Objective To recognize and identify the arginase(ARG)gene of Schistosoma japonicum(Sj),and to study its protection potential as a vaccine.Methods The 5'-end of the ARG gene from the Sj cercariae cDNA library was amplified by nested-PCR and the sequence was identified by bioinformatics.The complete coding sequence(CDS)was cloned into pET30a(+)vector,and a recombinant SjARG protein(rSjARG)was expressed,purified and used to raise antibodies.ARG's activity as an enzyme was tested by ornithine-ninhydrin reaction.Western blotting was used to compare the immunologic characteristics of rSjARG with that of the native one in Sj adult worm.Indirect immunofluorescence assay was used to immunolocalize it.For evaluating the protection potential of rSjARG,mice were immunized by the recombinant protein and challenged by cercariae of S.japonicum.Results The CDS length of the SjARG novel gene was identified as 1095bp.rSjARG showed enzyme activity and the same immunologic characteristics with the native arginase in adult worm.SjARG located in the genital organ and gut of both sexes.The worm reduction rate and egg reduction rate in rSjARG group were 55.8% and 48.8% respectively,higher than that of the rSj26GST group(28.6% and 6.89% respectively).Conclusion SjARG gene was identified,which shows a higher protection than the Sj26GST.