Table of Content

30 April 2004, Volume 22 Issue 2

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论著

Screening and Expression of Recombinant Human Monoclonal Antibody Fab Fragments Specific to Entamoeba histolytica

SHUQing;SHAOHong-xia;HiroshiTachibana;CHENGXun-jia

2004, 22(2):  1-69. 

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Objective To prepare recombinant human monoclonal antibody Fab fragments specific to the surface antigen of Entamoeba histolytica. Methods Total RNA was isolated from lymphocytes which were separated from an asymptomatic E. histolytica cyst carrier. The genes of IgG light chain and Fd region of heavy chain were amplified by a reverse transcriptase PCR and ligated with a plasmid vector. After the genes were introduced into Escherichia coli, the clones expressing Fab fragments specific to the surface antigen of E. histolytica were screened and the product was purified. Results Thirty thousand clones were screened and one of them was proved positive to the surface antigen of E. histolytica. Conclusion This study demonstrated that the bacterial system can be used to produce recombinant human monoclonal antibody Fab fragments specific to the surface antigen of E. histolytica and they may be applicable for the future diagnosis and treatment of the infection.

Effects of Tetracycline Operator Element Insertion on Plasmodium falciparum Glycophorin Binding Protein 130 Gene Promoter Activity

WANGXian-feng;XUECai-fang;LIUZhong-xiang;LIXun;LIShu-mei;LEIJun-chuan;MIAOJun

2004, 22(2):  2-75. 

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Objective To construct tetracycline operator (TetO) modified glycophorin binding protein 130 gene (GBP130) promoter of Plasmodium falciparum and investigate the position effect of insertion of TetO on the promoter activity. Methods Cloning of 7-copy of TetO (7cot) sequence into 4 points relative to transcriptional initiation site of GBP130 promoter in pGBPCATΔ2 plasmid (2 upstream and 2 downstream), respectively, produced 4 derivative plasmids, pG/7T(-5), pG/7T(-2), pG/7T(+2) and pG/7T(+5). After transient transfection, the expression level of reporter gene CAT in both pGBPCATΔ2 and its derivative plasmids was detected and analysed by CAT ELISA. Results Identification by enzyme restrictions and PCR amplifications, as well as DNA sequencing confirmed that the plasmids were successfully constructed. Transfection of these plasmids and CAT detection suggested that insertion of 7cot into each point of GBP130 promoter enhanced the promoter activity, and downstream location of 7cot in the promoter showed higher promoter activity than those located upstream, with the strongest effect in Ins 4 (point +5). Conclusion Plasmid pG/7T(+5), in which 7cot was inserted into +5 point of GBP130 promoter, can be chosen as the responsive plasmid in establishing tetracycline-controlled transgenic expression system of malarial parasites.

Expression of Proteinase Cathepsin L1 Gene of Schistosoma japonicum in Escherichia coli

YIBing;LIZhuo-ya;HEAi;ZHENGXiao-ying;ZHENGBin;ZHOUHao-jie;WANGYi;ZHANGRui-lin;YUNan;ZHANXi-mei

2004, 22(2):  3-79. 

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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 pGEX-4T-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-SjCL1was constructed. The expression product was detected by SDS-PAGE and Western blotting and an expression band about 62 000 was found. Conclusion The SjCL1 gene is effectively expressed in the E. coli JM109 cells.

Screening of cDNA Library of Schistosoma japonicum with Sera from Rabbits Vaccinated with Ultraviolet-attenuated Schistosomula

LIXiao-hong;LIUShu-xian;SONGGuang-cheng;XUYu-xin;CAOJian-ping;CHENJia-xu

2004, 22(2):  4-82. 

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Objective To search new potential schistosomiasis vaccine by screening cDNA library with sera of rabbits vaccinated with attenuated larvae. Methods Schistosoma japonicum (Sj) adult worm cDNA library was screened with sera of rabbits vaccinated with ultraviolet-attenuated schitosomula and by sera of infected rabbits, and the inserts of positive clones were amplified and sequenced. Results Six kinds of Sj genes were obtained after three rounds of screening. Among the genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine protease inhibitors (serpin), mitochondrion coding region, part of myosin heavy chain gene and two new genes, respectively. Conclusion Screening cDNA library with sera of animals vaccinated with attenuated larvae is an effective way to search new vaccine candidates for schistosomiasis.

Preparation and Identification of Monoclonal Antibodies against the Region II⁺ Motif in Circumsporozoite Protein of Plasmodium falciparum

ZHANGRui-juan;ZHUHuai-min;LIXiang-yu

2004, 22(2):  5-85. 

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Objective To develop and identify the monoclonal antibodies (McAbs) against Region II⁺ motif in circumsporozoite protein of Plasmodium falciparum. Methods BALB/c mice were immunized with 12 peptides within Region Ⅱ⁺ in circumsporozoite protein of P. falciparum. Spleen cells isolated from the immunized mice were fused with myeloma cell. After three times screening with ELISA, 3 positive hybridoma cell lines were obtained. Results ELISA test indicated that the McAbs reacted with recombinant circumsporozoite protein fragment containing tandemly repeat region and conserved Region II⁺.IFA test showed that the McAbs recognized not only the sporozoites of P. falciparum, but also the sporozoifes of P. yoelii. Conclusion McAbs obtained can probe the Region II⁺ motif in circumsporozoite protein of P. falciparum, which might also recognize that of other Plasmodium species.

Genotype Detection of the Merozoite Surface Protein Alleles of Plasmodium vivax

ZHANGShan-ying;XULong-shan;LUHui-min;ZHANGYing-zhen;GAOQi;LILi-sha

2004, 22(2):  6-89. 

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Objective To develop a method for detecting the genotype of Plasmodium vivax merozoite surface protein 1 (PvMSP-1) alleles. Methods According to the sequence characteristic of PvMSP-1, nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to amplify the polymorphic region of ICB5-ICB6 which contains Q repeats and PvuII restriction site (Sal-1 type). The PCR product was digested by PvuII restriction endonuclease and the digested fragments were observed by 2% agarose gel electrophoresis. The allelic type was determined according to the banding pattern. Results Bands in size of 400 bp (Belem type ) and/or 470 bp (Sal-1 type ) appeared in all 98 P. vivax isolates, no band was found in negative control. After PvuII digestion, two Sal-1 type fragments (120 bp and 350 bp) were obtained from 45 samples of 470 bp. Single-band of 400 bp appeared in 3 of 40 samples with 400 bp as Belem type, two bands of 120 bp and 280 bp appeared from other 35 samples as recombination type III, and another 2 bands with 120 bp and 240 bp as Korean isolate. Conclusion The result showed that the nested PCR-RFLP may be applied in the detection and identification of the three PvMSP-1 allelic types in China.

RAPD Analysis on Different Isolates of Trichomonas vaginalis

YUANLi-jie;GAOXing-zheng

2004, 22(2):  7-93. 

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Objective To study genetic polymorphism of DNA on seven isolates of Trichomonas vaginalis. Methods The random amplified polymorphic DNA (RAPD) technique was performed to amplify genomic DNA of the seven T. vaginalis isolates, including Beijing 1, Beijing 2, Chengde, Tangshan, Jiujiang 1, Jiujiang 2 and Jiujiang 3. The DNA bands detected were analyzed by clustering analysis with SPSS software. Results The percentage of genetic similarity among the seven isolates was from 77.4% to (94.7%,) showing a close genetic relationship among them. The percentages between the isolates of Beijing 1 and Tangshan, Jiujiang 1 and Jiujiang 2, Beijing 2 and Jiujiang 3 were 89.2%, 92.1% and 94.7% respectively, while that of Jiujiang1 and Chengde was 77.4%, indicating a lower homology. Conclusion There are a close genetic relationship and certain gene polymorphism among the seven T. vaginalis isolates; geographical origin plays little role to the genetic characteristics.

Soluble Expression of Plasmodium falciparum Glutamate Dehydrogenase in Escherichia coli, and its Purification and Identification

LIYan;NINGYun-shan;DONGWen-qi;LIMing

2004, 22(2):  8-97. 

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Objective To make soluble expression of Plasmodium falciparum (FCC1/HN) glutamate dehydrogenase(GDH) in Escherichia coli, purification and immunocompetence identification of the recombinant non-fusion GDH. Methods The GDH gene was cloned into prokaryotic expression vector pET23(a) to form recombinant expression vector pET23(a)/GDH. pET23(a)/GDH was transformed into E.coli BL21(DE3). Induced by IPTG(isopropyl-beta D-thiogalactoside), GDH was highly expressed in the supernatant after sonication. The soluble recombinant GDH was purified by Source-Q and Source-S chromatography. Enzyme-linked immunosorbent assay and Western blotting were carried out to identify the immunocompetence of the purified product. Results SDS-PAGE analysis showed that the soluble GDH protein accounted for approximately 15% of the total bacterial protein. By two-step ion-exchange chromatography, the purity of GDH reached more than 90% and the GDH possessed high antigenicity. Conclusion The soluble expression of GDH results in an integral three-dimensional structure epitope with high biological activity.

Application of EITB in Immunodiagnosis of Cysticercosis

]WANGLi-na;GELing-yun;MIAOFeng;YUZhen-hua;LIUYu-bing;WANGLi-na;GELing-yun;MIAOFeng;YUZhen-hua;LIUYu-bing;ZHENTian-min;LIGui-ping;YANGShu-fang

2004, 22(2):  9-100. 

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　Objective To establish a sensitive and specific method EITB for the immunodiagnosis of cysticercosis. Methods Two-dimensional electrophoresis was used to analyze and purify the cyst fluid antigen. Western blotting using the sera of patients with cysticercosis, hydatidosis, and other heteroserum was used to screen the specific antigens. EITB using the specific antigen was established and compared with ELISA. The detection effect of serum and blood on filter paper in EITB was also compared. Results Two specific antigens with isoelectric point (pI) of 9.4 and Mr of 14 000 and 16 600 were obtained, respectively. EITB method based on these antigens was established with the sensitivity and specificity of 92.5% and 100%, respectively, significantly higher than that of ELISA. The sensitivity of serum and filter paper blood was similar in EITB. Conclusion A sensitive and specific EITB method for immunodiagnosis of cysticercosis was established.


Ultrastructural Study on Pharyngeal Armatures of Seven Species of Sandflies in China by Scanning Electron Microscopy

GUODong-xing;JINChang-fa;HONGYu-mei;NIBing;QIAOZhong-dong

2004, 22(2):  10-102. 

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　Objective To observe the ultrastructure of pharyngeal armature of 7 species of sandflies in China. Methods The pharyngeal armatures of various sandflies were studied by scanning electron microscopy. Results The pharyngeal armature of sandfly consisted of pointed-teeth with various shape, number and arrangement among different species. Conclusion Such differences may provide the morphological proof for identification of species.


实验报道

Screening of Mimic Epitopes of Trichinella spiralis Antigen from Phage 12-mer Peptide Library

PANHong;JIANGChang-fu;LITian-qun;LEIJia-hui;SHIHong-bo;WEILan-ying

2004, 22(2):  11-105. 

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　Objective To screen special mimic epitopes of Trichinella spiralis antigen from peptide library for exploring new diagnostic antigens. Methods Ts-IgG purified from serum of trichinosis patients was used to screen the phage 12-mer peptide library for 5 rounds. 24 clones were picked out randomly to detect the immunoactivity. The sensitivity and specificity of the 6 clones (T1-T6) whose A values were higher than others were tested by ELISA. Results The sensitivity of the clones T1-T6 was the same with larval antigen of Trichinella spiralis (TsA) (positive rate: 100%, P>0.05), and there was no difference in specificity between T1-T6 and TsA (negative rate: 0-40%, P>0.05); T3 and T6 did not react with sera from patients of paragonimiasis, showing higher specificity than TsA(P<0.05); T6 did not react with sera from patients of schistosomiasis, also showing higher specificity than TsA(P<0.05). Conclusion The mimic antigenic epitopes of Trichinella spiralis have been successfully obtained by screening phage 12-mer peptide library.


Detection of Biliverdin Reductase Activity of Schistosoma japonicum

LIUWen-qi;LIYong-long

2004, 22(2):  12-108. 

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　Objective To explore the presence of biliverdin reductase (BR) activity in adult worms of Schistosoma japonicum in vitro. Methods The soluble fraction was isolated from homogenates of adult worms of S. japonicum, and incubated with biliverdin under different pH conditions and buffers. The time dependency of this enzymatic reaction was also detected. ResultsThe soluble fraction of the homogenates of adult worms could degrade biliverdin in vitro, the BR activity was 43.30 nmol/(mg·min), with its optimal condition at pH 8.7. The rate of reaction peaked at 15 min of incubation for the BR activity. Conclusion The presence of BR activity in adult worms of S. japonicum was firstly demonstrated.


学术争鸣

Studies on Paragonimus proliferus

ZHOUBen-jiang

2004, 22(2):  13-112. 

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　Objective To make identification between Paragonimus menglaensis and Paragonimus proliferus. Methods Crabs were collected from same area where P. proliferus and P. menglaensi were reported, metacercariae and excysted metacercariae were separated. Adult worms were collected from experimental infection and identified. Results The metacercaria is large, with an average size of (1.23±0.087)mm×(1.10±0.073)mm, covered with a thin and fragile cyst wall; the size of excysted metacercariae is (2.01±0.71)mm×((0.62)±0.12)mm, with irregular bough-like wrinkles excretory bladder resembling in front of ventral sucker, two pointed and slim distal ends of gut locate at 1/6 of the body from the tail end; the adult worm has large uterine mass, with an average length of 1/(4.2) of the whole body. The natural definitive host for P. proliferus is not monkeys, dogs, and cats, but rats. The metacercaria of the reported P. menglaensis has been mixed up with that of P. microrchis from the same crab, excysted metacercaria has been same to that of P. proliferus, and an immature worm has been mistakenly identified as its adult worm. Conclusion P. proliferus is a valid independent species, while P. menglaensis is a mis-identified, invalid one.


专家论坛

Classification of the Sand-mite Family Walchiidae (Acariformes: Trombiculoidea)

WENTing-huan

2004, 22(2):  14-118. 

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　The priority of Walchiinae Ewing, 1946 based on Walchia Ewing, 1931 or Gahrliepiinae Womersley, 1952 based on Gahrliepia Oudemans, 1912 as Walchia regarded as subgeneric taxon had been a controversy for almost half a century since 1952. Both genera are valid now. Wen (1999) redefined both subfamilial characters and in turn promoted Walchiinae to a full (fami)lial status. Walchiidae (Ewing,1946) Wen, 1999 is characterized by SIF=4B/4Bs/5B/6B-N/B-3/2-2(1)1(0)1(0)0.0000, small to large sized sand-mites, IP=320-1220. Scutum is small to large size, extending backward over part of dorsum, and pentagonal with acuminate posterior angle or tongue-shaped. The scutum is never provided with anteromedian setae (AM or vi) and anteromedian projection(A or N=0). Scutal setae have AL and PL pairs basically, frequently in addition with 2-40 accessories (PPLs), and rarely 1-2 pairs of intermedial setae (IM). Sensillae (Sn or sci) are short and expanded. Leg segments are 7.6.6 always without variations.Casting off the anteromedian setae on scutum, increasing the leg segments and reducing the tactile body setae are the plesiomorphic characters of sand-mites, that means Walchiidae in higher advance of evolution than both Trombiculidae (Ewing, 1929) and Leeuwenhoekiidae (Womersley,1944). It is rationally to unify three families of vertebrate parasitic larvae into a single superfamily, Trombiculoidea nec Welbourn (1991), that separable from superfamily Trombidioidea of arthropod parasitic larvae and standing at most advanced evolution of Parasitengona.Family Walchiidae has 2 subfamilies, Walchiinae Ewing, 1946 sensu Wen 1999 and Gahrliepiinae Womersley, 1952, sensu Wen 1999. Each subfamily contains two tribes, Walchiini (Ewing, 1946) Wen 1984 and Schoengastiellini Wen 1984 for Walchiinae, and Gahrliepiini sensu Wen 1984 and Intermedialiini Wen, 1984 for Gahrliepiinae. Currently this family has 18 genera and 28 subgenera, 248 nominated species and subspecies. Walchiid sand-mites are essentially an Old World family and best developed in the Oriental Region with the center of development in Southeast Asia.