Table of Content

30 October 2002, Volume 20 Issue 5

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论著

Comparative Study on the Characteristics of Immune Response Induced by 97kDa DNA and UV-Attenuated Cercariae Vaccines of Schistosoma japonicum

CHENJiaxu;LIUShuxian;CAOJianping;SONGGuangcheng;XUYuxing

2002, 20(5):  1-261. 

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　Objective To compare the protective immunity against challenge infection and the characteristics of immune response induced by nucleic acid vaccine encoding full length paramyosin of Chinese S. japonicum (Sjc97 DNA ) and ultraviolet attenuated cercariae vaccine(UVC). Methods C57BL/6 mice were vaccinated intramuscularly with the Sjc97 DNA twice at interval of wk 0 and wk 3, and challenged in three weeks after immunization with Sjc97 DNA (Sjc97 DNA group). C57BL/6 mice in UVC group were challenged with non attenuated cercariae five weeks post immunization with UVC. Another two groups of mice were vaccinated with blank plasmid and infected with non attenuated cercariae without immunization respectively to serve as control. Seven weeks after challenge infection the mice were perfused to obtain worm burden and the number of eggs in the liver tissue. The specific anti SEA and AWA IgG, IgA and IgG subclasses in sera of the mice and cytokines secreted by splenocytes exposed to mitogen Con A for 96 h at pro and post challenge were determined by ELISA test. Results C57BL/6 mice immunized with Sjc97 DNA or UVC before challenge infection produced predominantly cytokines of IL 2, IFN γ and antibodies of IgG2a, IgG2b and IgA. The levels of above cytokines and antibodies were higher in UVC group significantly than that in Sjc97 DNA group. The mice immunized with Sjc97 DNA showed a worm reduction rate of 36.3 % and egg reduction rate of 42.4 % in the liver, and in the UVC group, 66.9 % worm reduction rate and 75.6 % egg reduction rate in the liver. At the seventh week after challenge infection with non attenuated cercariae, the immune responses of Th1 type in the mice of two vaccine groups were still predominant, although Th2 type responses increased slightly; and Th2 type responses of the mice in the blank vector and infection control groups were predominant. Conclusion The nucleic acid vaccine, Sjc97 DNA, and UV attenuated cercariae could all induce protective immunity in C57BL/6 mice significantly. The protective immunity induced by UVC is stronger significantly than that by Sjc97 DNA, and is obviously correlated with Th1 type responses.


Construction and Expression of a TRAP/CSP Chimeric Protein of Plasmodium falciparum

DUJingling;PANWeiqing;QIANFeng;XIEChao

2002, 20(5):  2-265. 

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　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.


Cloning and Expression of a Secretory Protein Sjsp16 of Schistosoma japonicum

HUWei;LIUFeng;WANGJujun;XUXuenian;HANZeguang;FENGZheng

2002, 20(5):  3-269. 

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　Objective To clone and express a new secretory protein Sjsp16 of Schistosoma japonicum. Methods The specific primers were designed according to the EST sequence and used for amplification of the encoding sequence from the cDNA clone containing Sjsp16. The gene was subcloned into pET28 plasmid and expressed. The structure and functions of this protein were analysed by bioinformatics method. Results The secretory protein gene of Schistosoma japonicum Sjsp16 was cloned and expressed successfully. The bioinformatics analysis showed that there is a signal peptide at the N terminal, which is the character of a secretory protein. It is predicted that Sjsp16 protein contains ML domain, one N glycosylation site, three phosphorylation sites for protein kinase C, four phosphorylation sites for casein kinase II and three N myristoylation sites. Conclusion The protein encoded by Sjsp16 is a secretory one with lipid recognition and lipid binding function, and might be a new candidate for drug target or vaccine.


Immunoscreening and Sequence Analysis of a cDNA Library of Adult Trichinella spiralis

YANGYaping;ZHUXinping;YANGJing;ZHOULei;HUANGSong

2002, 20(5):  4-273. 

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　Objective To obtain an antigenic gene of adult Trichinella spiralis. Methods cDNA library of the adult Trichinella spiralis was screened using the sera of immunized and infected rabbits. The gene sequence was analyzed by DNAstar software and GenBank database. Results Nine positive clones were identified by immunoscreening. The clone Ts87 was sequenced and a cDNA with 1 172 bp full length was obtained using 5′ RACE technique, encoding 347 amino acids. Some possible antigen epitopes were predicted. Conclusion A novel antigenic gene of Trichinella spiralis was obtained.


Cloning and Expression of the CryIVD Gene of Bacillus thuringiensis subsp. israelensis and its Mosquito Larvicidal Activity

ZHANGXin;LIUXiangping;YANGe;ZHENTianmin#;WANGXinguo;SUNChuanhong;WANGHuaiwei

2002, 20(5):  5-277. 

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　Objective To clone and express Bacillus thuringiensis subsp. isrealensis(B.t.i.) crystal protein CryIVD gene and determine its mosquito larvicidal activity. Methods The gene encoding CryIVD (2.0 kb or so) was amplified by PCR, the amplified fragment was inserted into E.coli plasmid pUC18 to construct the recombinant cloning and expression vector pUC18 CryIVD, which was named pUC18 1. The ligation was transformed into competent E.coli DH 5α and the recombinant vector pUC18 1 was confirmed by restriction enzyme digestion and DNA sequencing. After being inducted by IPTG, the expression of CryIVD gene in positive clone was detected by SDS PAGE and the mosquito larvicidal activity of CryIVD was also determined by standard bioassay. Results The results showed that the CryIVD gene was successfully cloned and expressed in E.coli DH 5α . Mosquito larvicidal activity of engineered E.coli (LC 50 ) to Cx.pipiens pallens and Ae.albopictus Ⅱ-Ⅲ instar larvae was 2.38×10 6 cells/ml and 1.6×10 7cells/ml respectively. Conclusion The CryIVD gene was successfully cloned and expressed, and a high mosquito larvicidal activity was observed.


Cloning and Expression of the Antigen Structural Gene TspE1 from Pre-encysted Larvae of Trichinella spiralis

CUIJing;WANGZhongquan;WANGHuizhi;ZHAOGuoqiang;ZHANGHongwei

2002, 20(5):  6-280. 

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　Objective To clone and express the structural gene encoding a 31 kDa antigen of Trichinella spiralis (Henan isolate) pre encysted larvae (TspE1). Methods On the Day 17 after being infected with Trichinella spiralis ,pre encysted larvae were collected and total RNA of the larvae was obtained.The target gene in the recombinant plasmid (pUC18/Ts HN3) was sub cloned into the prokaryotic expression vector pGEMEX 1 and the recombinant pGEMEX 1/Ts HN3 was constructed. After IPTG induced incubation, the fusion protein was expressed in E.coli JM109(DE 3)competent cells, analysed by SDS PAGE and identified by Western blotting. Results The results of SDS PAGE demonstrated that the target gene was efficiently expressed and the level of expression peaked at 4 h post incubation. The molecular weight of the recombinant protein was 31 kDa. The portion of the fusion protein accounted for 26% of all the proteins by thin layer gel optical scanning. The fusion protein could be recognized by sera from rats infected with Trichinella spiralis and from patients with trichinellosis. Conclusion The gene encoding a 31 kDa antigen of Trichinella spiralis ( Henan isolate) pre encysted larvae ( TspE1) was cloned and expressed successfully in prokaryotic vector.


Construction of Cryptosporidium parvum cDNA Library and Cloning of P23、CP15/60

XUWeidong;ZHANGXichen;KONGQingchang;LIUMingyuan;YINJigang;LIJianhua;YANGJu;HEHongxuan;LYajian

2002, 20(5):  7-284. 

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　Objective To study the genetic background of Cryptosporidium parvum local strain and to provide a DNA source of the parasite. Methods With Promega′s kits, total RNAs and mRNAs were isolated from C. parvum and cDNAs were synthesized by reverse transcription. A cDNA library was prepared with pUC18 vector and host cells of E. coli strain DH5 alpha. Two PCR primers were designed and synthesized to screen protective genes. PCR products were cloned and sequenced. Results There are 1.9×10 6 recombinants in this library. The cDNA fragments ranged between 0.4×10 3 bp and 6.5×10 3 bp. Two genes encoding 23 kDa and 15/60 kDa sporozoite proteins of C. parvum respectively from the library were cloned and sequenced. Conclusion A cDNA library of Cryptosporidium parvum was prepared successfully.


The Role of IFN-γ and IL-4 in Defending Pneumocystis cariniiInfection

ANChunli;WANGJichun;WANGXuelian

2002, 20(5):  8-288. 

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　Objective To investigate the role of cytokines interferon gamma (IFN γ) and interleukin 4 (IL 4) in host defense to Pneumocystis carinii infection. Method Two different murine models (IFN γ or IL 4 gene knockout C57BL/6 mice,15 mice each strain) were contagiously infected with P.carinii by cohousing with P.carinii positive mice for 3,5,7 weeks as well as 15 gene un knockout C57BL/6 mice for infection control group. Another 15 non contagious C57BL/6 mice were as the normal control group. The number of P.carinii ,the CD4 + and CD8 + T cells, and specific IgG were detected. Results The number of P.carinii differed in different groups of mice cohabited with P.carinii infected mice. The number of the organisms in the lungs of IFN γ knockout mice increased significantly ( P < 0.05),showed a slight increase in the lungs of IL 4 knockout mice as well as the gene un knockout mice ( P > 0.05). The CD4 +and CD8 + T cells response was in parallel to the burden of cysts in the lungs but specific lgG increased gradually in 3 groups after co housing. Conclusion IFN γ is important in host for defense from P.carini infection. Absence of IL 4 does not affect the resistance of mice against P.carinii infection.


The Diagnostic Value of ELISA in Detecting Specific IgG_4 of Clonorchiasis

ZHANGShunke;ZENGXianfang;YIXinyuan;LIZhongjie;CAIChun;TIANMingli;ZHANGZuping;SHENJie

2002, 20(5):  9-291. 

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　Objective To prove the value of ELISA in detecting serum IgG 4 in the diagnosis of clonorchiasis. Methods ELISA was used to detect the specific IgG and IgG 4 antibodies in sera from the patients with clonorchiasis and other parasitic diseases and from healthy persons. Results The sensitivity and specificity of IgG 4 antibodies detected by ELISA in sera from the patients with clonorchiasis showed no significant difference with that of IgG detected. The cross reaction rate of IgG 4 in sera from patients infected with other parasites was significantly lower than that of IgG detection. The diagnostic efficiency and the positive and negative predictive values were 96.45%, 100% and 92.85%, respectively. Conclusion The specific IgG 4 detected by ELISA showed the same sensitivity and specificity as IgG in diagnosing clonorchiasis and less cross reaction with sera from the patients infected with other parasites. Therefore, the specific IgG 4 detection by ELISA had higher diagnostic value than IgG detection in diagnosing clonorchiasis.


Immune Responses to Challenge Infection in Mice Immunized with Trichinella spiralis Adult Worm Soluble Antigen

SHENLijie;LUOZhiyong;ZHUShenghua

2002, 20(5):  10-294. 

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　Objective To study the immune response induced by the mice immunized with Trichinella spiralis(T.spiralis) adult worm soluble antigen(AWSAg). Methods T.spiralis AWSAg was prepared to immunize Kunming strain mice. Dynamic changes of IgG, IL 2 and T lymphocyte subsets from immunized mice were determined after challenge infection on d 7, d 14 , d 21 , d 28 and d 35 . Results On d 7 after challenge infection, IgG and CD4 + T cells of immunized group were markedly elevated and persisted higher over the observation period. In contrast, CD4 + T cells and CD4/CD8 ratio were significantly decreased in unimmunized group resulted from immune suppression after infection. IL 2 levels reached the peak on d 7 and persisted in high level from d 7-d 21 in both immunized and unimmunized group after infection, then decreased gradually. Till 35 days after infection, IL 2 level was still higher than the normal mice. Conclusion Mice immunized with AWSAg of T. spiralis produced a potential cellular and humoral immune response.


实验报道

Comparative Study on Genomic DNA of Malaria Parasite: Chloroquine-resistant and Chloroquine-sensitive Strains of Plasmodium berghei

CHENKeqiang;SONGGuanhong;ZHUHuaimin;XinZhuanSU

2002, 20(5):  11-297. 

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　Objective To investigate the genomic DNA difference between chloroquine sensitive (N) and chloroquine resistant (RC) strains of Plasmodium berghei . Methods By means of polymerase chain reaction(PCR), the abstracted genomic DNAs of the N and the RC strains of Plasmodium berghei were amplified with 37 random primers(RAPD) and 84 anchored primers (APAD). The PCR products were analysed by electrophoresis and statistics. Results Of 440 bands amplified with RAPD, 196 bands were common to both strains whereas 43 bands were unique to either the RC or the N strain, with a similarity coefficient (SI) of 0.89, and a distance coefficient (D) of 0.197. Similarly, among 952 bands amplified with APAD, 436 bands were shared by both strains, and 53 bands were unique to either of them with SI 0.91 and D 0.155 respectively. Conclusion The genomic DNAs between the RC and the N strains of Plasmodium berghei are highly homogeneous.


Identification of Metalloproteinase in Intestinal Contents Of Adult Schistosoma japonicum

YUANShishan;YIXinyuan*;ZENGXianfang;ZHANGShunke;HUANGyuelong;LarryMcReynolds

2002, 20(5):  12-301. 

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　Objective To identify the proteinases in intestinal contents of Schistosoma japonicum (S.japonicum) adult worms. Methods Proteinases were fractionated through gelatin SDS PAGE, and gelatin was degraded by proteinases when the gel was incubated in buffer with optimal pH. Metalloproteinase which degrades gelatin was identified by incubating gel in optional buffer containing different enzyme inhibitors. Metalloproteinase was isolated through SDS PAGE. Results A metalloproteinase was identified in intestinal contents of S.japonicum adult worms. The optimal pH of metalloproteinase is pH 7-9. The activity of the metalloproteinase was inhibited by EDTA. The metalloproteinase showed enzymatic activity after being fractionated through SDS PAGE and weakly reacted with sera of rabbits infected with S.japonicum . Conclusion A metalloproteinase is present in intestinal contents of S.japonicum adult worms.


Effect of Cyclosporin A to T Lymphocyte Subsets and Toxoplasmosis After Heart Allotransplantation in Rat

HANHui;ZHONGHongxing;ZHANGYongshang

2002, 20(5):  13-304. 

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　Objective To evaluate the effect of Toxoplasma gondii infection on immune function of the rat recipients and onset of Toxoplasmosis after heart transplantation and its correlation with the use of Cyclosporin A(CsA). Methods ELISA was used to detect recipient's specific circulating antigen (CAg) and antibodies (IgG, IgM) after the transplantation. T lymphocyte subsets in peripheral blood were examined by using immunofluorescence stain and flow cytometry (FCM) before and after heart allograft 5,10,15,20 days in rats. Results The use of CsA increased the risk of infection by T. gondii and accelerated the increase of CD8 + T lymphocyte after the transplantation. The incidence of donor acquired T.gondii infection was higher than that of reactivated silent infection in recipients before operation. The percentage of CD8 + T lymphocyte was evidently elevated due to the onset of toxoplasmosis and the ratio CD4 +/CD8 + was reduced or inverted in the meanwhile. Conclusion The immune suppression after use of CsA was the main reason leading to an activation of the silent infection of T.gondii . CD8 + was the main cytotoxic cell elevated during the infection.


Development of Taenia Solium Cysticercus and Morphological Observation

LIUYongjie;LIQingzhang;HAOYanhong

2002, 20(5):  14-307. 

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　Objective To systematically study the development and morphological changes of Taenia solium cysticerci in the period from the appearance to maturation. Methods Pigs were orally infected with T.solium eggs. Autopsies were made periodically to get tissue specimens and examine the distribution of cysticerci. Cysticerci were excised, pressed between two microscopic slides for microscopical observation. Sections were also made for histological study. Results Cysticerci distributed mainly in muscles, secondly in brain, liver, lung and kidney. The size of cysticerci was highly variable at different parts or even in the same part of the same host. The early stage cysticercus was observed on the 19th day without suckers and hooklets on the scolex region, and was only found in skeletal muscles. Histological examination revealed that early development of the scolex became obvious. No cysticerci were found in other tissues. At 30 d, the cysticerci possessed hooklets and brood suckers. With a longer duration of infection, the diameter of suckers and length of hooklets both increased. Larger suckers and more folds on scolex appeared 60 d later. Morphologically, cysticerci after 60 d were similar to those found at 60 d. Degenerated cysticerci were found at every stage. Conclusion Cysticerci appear in about 19 days after the pig was infected with eggs of T.solium and the duration needed for maturation is about 60 d.


Observation on Decline Phase of Maintenance of Brugia malayiMeriones unguiculatus Model

CHENShaohong;SUNDejian;SHIHenghua;YUANYizhen

2002, 20(5):  15-309. 

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　Objective To observe the degeneration of Brugia malayi in Meriones unguiculatus model. Methods Microfilaria of Brugia malayi derived from Meriones unguiculatus was used to infect Anopheles sinensis . Infective stage larvae (L 3) from mosquito vector were collected and inoculated into abdomen of Meriones unguiculatus. Successive 33 generations of the parasite in the rodent model have been observed. Results Since 1974 when the animal model was established, the parasite has lasted for 33 generations, the positive rate of Meriones unguiculatus with microfilaria gradually reduced from 80% of the 28th generation to 16% of the 32nd generation and finally to 0 at the 33rd generation. Conclusion It became difficult for the larvae of Brugia malayi to develop and/or reproduce in the animal model after multiple inoculations for generations.