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The Study on a Vaccine Against Schistosomiasis

Author: ZhuChuanGang
Tutor: LinJiaoJiao
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: Schistosoma japonicum vaccine Cholera toxin B subunit Gynecophoral canal protein SJ.28GST Sj.23kd HBcAg gene expression profile
CLC: R392
Type: PhD thesis
Year: 2006
Downloads: 331
Quote: 2
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Abstract


Schistosomiasis is an important parasitic disease which threatens severely the health and life of human beings. Nevertheless, the current control strategies seem impossible to control schistosomiasis and its transmission effectively. Therefore, the new strategy of control measure by vaccination has been paid more attention. Our research focus mostly on the way to develop an effective vaccine as a tool for schistosomiasis control: study on the construction of mucosal vaccine and its effect, construction of recombinant transfer vector inserting peptide epitope of schistosoma japonicum into hepatitis B core antigen, study on the effect of cocktail vaccines against schistosomiasis. Meanwhile, study on the transcriptome profiling in the gender development of schistosoma japonicum to seek for the new vaccine-candidate molecules.1. Construction, Expression and immune protection of epitopes of Sj. 28GST fusion with cholera Toxin B SubunitCholera toxin B subunit(CTB) is considered to be an effective adjuvant and carrier protein. The expression plasmid pET30(a)-CTB was constructed firstly, then epitopes of Sj. 28GST was fused and expressed with CTB and its protective effect was examined. In order to express ctb gene in E. coli, The expression plasmid pET30(a)-CTB was constructed by inserting the ctb gene into the plasmid of pET-30a(+) of Novagen with a 6×His tags in the 3’end, then it was transformed into E. coli BL21(DE3) and was induced by IPTG. SDS-PAGE analysis revealed that the molecular weight of this expressed product was about 14kD. The recombinant protein was expressed as inclusion body. After being denatured, the recombinant protein was purified by His bind column. Western Blot showed that the recombinant protein could be recognized by CT antibodies, indicating that this expressed product had good antigenicity of CTB. Its biological activity was also conformed by GM1-ELISA that the fusion protein can bind to ganglioside(GMI).To constructed plasmid pETCTB-SJ. 28GST containing CTB and epitopes of SJ. 28GST gene and expressed it in E. coli. According to the amino acid sequence of the Sj. 28GST, we deduced the DNA sequences of its epitopes and then synthesized chemically four of them. We fused them in frame with cholera toxin B subunit to get pETCTB-SJ. 28GST. After expressing in E. coli BL21(DE3), their properties were analyzed. The study demonstrated that the recombinant plasmid could express the CTB and Sj. 28GST fusion protein in form of inclusion body in E. coli. The CTB-Sj. 28GST fusion protein could be recognized by anti-serum in western blot. Then, Mice were divided into 10 groups each with 10 mice. They were immunized intragastically and under skin respectively with rSj. 28GST, rCTB, rSj. 28GST+rCTB and rCTB-Sj. 28GST, PBS was used as control groups. The mice were challenged with 40±1 S.j cercariae per mouse lwk after the fourth vaccine. Forty-five days later, mice were killed and perfused, and the adult worms and eggs were counted. The worm reduction rate of rCTB-Sj.28GST was 28.6%and 17.7%in the intragastric vaccination groups and in the skin vaccination groups respectively. The egg reduction rate was 46.3%and 21.3%respectively. This study showed that a significant immuneprotection against Schistosoma japonicum infection was induced by mucosal vaccination with rCTB-Sj.28GST.2. Clone and Expression conditions of the gene Encoding Gynecophoral Canal Protein ofSchistosoma japonicumGynecophoral canal protein of Schistosoma japonicum(Sj. GCP) is a highly expressed gene in male and may be implicated in the sexual development of female schistosomes and or egg production. According to the sequence of the ORF of the target gene Sj. GCP, a pair of primers, in which XholI and HindⅢsites were taken into account respectively, were designed and used to amplify the target gene using SjGCP/pGME-T as template. Then, subcIoned the gene into plasmid vector pGEX-6p-1 and induced by IPTG for its expression. The recombinant SjGCp/pGEX-6p-1/Ecoli BL21 was induced under different conditions. The results showed that the inserted gene was 1950bp length, with a ORF of 1932bp which encoded 643 amino acids. The deduced molecular weight of the protein was 62kDa. At the concentrations of range from 0.01 mmol/L-2.0mmol/L IPTG, at the temperature ranging from 15℃-37℃and in the duration of 0.5-16 hour, the amount of the soluble protein in the supernatants were almost not increased, which indicate that the fusion protein was surely existed in the form of inclusion body. Another strategy was used to purify the fusion protein by the procedures of washing and separation-degeneration-regeneration combined with protein chromatography. At the same time, the DNA vaccine, pcMV-Sj. GCP, was constructed.3. Study on the cocktail vaccines and their effection against schistosomiasisTo test the cooperation effect of cocktail vaccines in immune protection against challenge infection in mice, three recombinant proteins: glutathione S-transferase(rSj.28GST), 23kDa membrane protein large hydrophilic domain (rSj23), and rSjGCP and their recombinant DNA vaccines (VR1012-Sj.28GST, VR1012-LHD-Sj23, pcMV-rSjGCP )was used. The recombinant antigen rSj28GST was used to explore the optimal immune times. Mice were divided into 13 groups each with 10 mice. They were immunized with difference vaccines plus adjuvant 206 respectively. PBS and adjuvant 206 were used as control groups. The mice were challenged with 40±1 S.j cercariae per mouse after the third vaccinization. 6 weeks later, mice were killed and perfused, the adult worms and eggs were counted. Results, the worm reduction rate was different from 6%to 39%, the egg reduction rate was from 24%to 47%. The cocktail DNA vaccine get the best protection effect against the infection in all these groups with 39%(P<0.05) worm reduction rate and 47%(P<0.05) egg reduction rate. Cocktail vaccines of three or two recombinant proteins have induced immune protection against schistosomiasis as they were used respectively, but there is no significant difference. The rSj. 28GST that vaccined 3 times can get more effect against challenge infection than the 2 times or 1 time group, but there are no significant too.4. Cloning and construction of recombinant transfer vector inserting peptide epitope of schistosoma japonicum into hepatitis B core antigenHepatitis B virus core antigen(HbcAg) is a more useful protein that can enhance the immunogenicity of peptide epitope. HbcAg gene was amplified by polymerase chain reaction(PCR) and the site-directed mutate genesis was used to mutate the HbcAg at c/el 80-81 amino acid site to SalI restriction endonucleases site, GTCGAC. The PCR product was digested with restriction endonucleases, NcoI and SacI, and inserted into the vector pBacPAK-His1 to construct the transfer vector, pBacPAK-His-HbcAg. Then, the immunogenic epitopes genes of 3 schistosomia japonicum genes, Sj. GCP, Sj. 28GST and Sj. 23kd were fused to hepatitis B virus core antigen at c/el 80-81 amino acid site respectively, 3 recombinant transfer vectors were constructed successfully. The result of nucleotide sequencing showed that cloned target genes had positive reading frame in the plasmid. This work provides a new way to develop schistosomia vaccine.5. Transcriptome profiling study in the gender development of schistosoma japonicumSeeking for the new vaccine candidate molecules is also the important aspects of the vaccine research work. To identify genes, cDNA microarray was used to show globally measure changes in mRNA expression levels during the gender development, cDNA microarrays of Schistosoma japonicum, which cDNA derived from SSH libraries, representing 1536 female and 1536 male cDNA clones, were constructed and used to screen RNA from schistosomulum(14, 17, 20, 24 days) to adult worm(28, 42 days), adult male and female worm(42days). The permanents of the reliability and the reproducibility of the microarray data were analyzed by correlation coefficient(R), consistence rate (CR). the correlation coefficient of data from this cDNA microarray system was more than 0.9 and Consistence rate was 100%. The result proves the accuracy of the cDNA microarray data.Microarray hybridization results revealed that there were 1372 female and 1102 male differentially expression gene clones. 1117 gene clones were selected and sequenced. The results of sequence analysis indicated that these gene clones represented 562 difference genes, among them, 206 genes highly expressed in male, 256 in female and 100 in both male and female. Homology comparison of sequenced ESTs were analyzed and functions of genes encoded protein were predicted. Clustering of genes by expression profile across the development stages revealed a number of up-regulated genes at different stage,The microarray result was verified by Real-time PCR, which demonstrated that the gene was indeed differentially expressed, and the high consistence rate(0.94) confirmed the cDNA microarray results, thereby supporting the reliability of the system.Our result are useful in understanding the molecular mechanisms regulating schistosome sex developmental processes and to better understanding schistosome special mechanism in growth, replication and interaction between male and female on molecular level, as well as in seeking for the new vaccine candidate molecules..

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