Dissertation > Medicine, health > Basic Medical > Medical Immunology > Immunobiology

Preliminary Study on Subunit Vaccine and Nucleic Acid Vaccine of Echinococcus Multilocularis

Author WangChangYuan
Tutor ChenYaTang
School Chongqing Medical University
Course Infectious Diseases
Keywords The Echinococcus tapeworm elp gene Subunit vaccine DNA vaccines Immune response BALB / c mice
CLC R392.1
Type PhD thesis
Year 2003
Downloads 126
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Objective To construct a multi-Echinococcus elp gene of prokaryotic expression vector and expressed in E. coli and purified recombinant protein; build elp gene eukaryotic expression vector, to verify the ability of the expression and purification of recombinant plasmid transient expression in mammalian cells COS7; prokaryotic expression of recombinant protein subunit vaccines, animal eukaryotic expression plasmid DNA vaccine, the immune response generated by the observation of experimental animals; compare two kinds of vaccine and the effect and Freund's adjuvant subunit vaccine and mouse IL-12 eukaryotic expression plasmid multi Echinococcus DNA vaccine adjuvant effect. Artificially infected gerbils, collecting more than Echinococcus metacestode larvae (EMML). Nucleic acid purification kit total RNA extraction EMML. Obtained from the GenBank the Echinococcus tapeworm elp gene cDNA sequence (EM10 EM II / 3), to design synthetic PCR primers and the introduction of the endo enzyme sites, to prepare the desired gene from EMML RNA by RT-PCR method. At the same time, compared the effect of nucleic acid extraction kit RNA was prepared and DNA polymerase amplification of long DNA. Application of molecular cloning technology, the RT-PCR amplification products were inserted the cloning vector the pGEM-11zf and sequencing and sequence analysis. Subcloned containing application the Echinococcus tapeworm elp gene of the complete coding region of the DNA sequence subcloned in the prokaryotic expression vector pET32a () and pQE30 () of. Induced by IPTG, Western blot identification. Subcloned in the eukaryotic expression vector pcDNA3.1 (). Elp gene eukaryotic expression recombinant plasmids for mammalian cells transfected COS7. RT-PCR, COS7 cells elp lt; WP = 14 gt; gene transcripts, Dot-ELISA and immunohistochemistry identified elp gene product. Large amounts of purified the ELP recombinant protein expressed in E. coli and elp gene eukaryotic expression plasmid. 72 4-6w age of BALB / c mice by male and female half divided into 7 groups (10 6 / group, and finally a group of 12). A group of: each rat the ELP recombinant protein 100 ug; Group B: Each rat with ELP recombinant proteins 100μg Freund's adjuvant mixed with an equal volume (Initial Freund's complete adjuvant, the second three incomplete Freund's adjuvant) group I:; NS groups: saline control; each mouse with a eukaryotic expression empty vector (pcDNA3.1 ()) 100 ug, 0.75% bupivacaine 480μl; Ⅱ group: mice with elp gene of eukaryotic expression recombinant plasmid ( -pcD-ELP) 100μg, bupivacaine 480μl; group III: each mouse with mouse IL-12 eukaryotic expression recombinant plasmid (PIL-12) 100 ug, bupivacaine 480μl; IV Group: each mouse PCD-ELP Pil-12 plasmid 100 ug 100 ug, bupivacaine 480μl. A group and B group multi-point subcutaneous injection; NS group, group I, group II, group III and group IV with NS adjusted to a final volume of 140μl, bilateral quadriceps intramuscular injection (70μl each side). Each group were immunized 3 times at an interval 2w. Detected by ELISA before immunization (0w) and at different times after immunization (after the first immunization, 2w, 4w, 6w, 7w)-specific IgG1, IgG2a and IgG2b levels. Double-antibody sandwich ELISA test kit after the first immunization 7w each group of mice spleen mononuclear cells (PMNC), by specific antigen stimulation, produce IL-4, IL-12 and IFN-gamma ability to 3H-thymidine incorporation assay spleen lymphocytes value-added. Results 5 kinds of commercially available kit or reagent, total RNA extraction kit (RNeasy Total RNA Kit) effect. Only the kit to extract EMML RNA electrophoresis showed a clear 28 S, 18 S rRNA bands and dispersion 0.8kb to 20kb undegraded mRNA, is the only way to reverse transcription single target band of amplified nucleic acid extraction thereof. Taq DNA polymerase fidelity DNA polymerase Pfu the mixed formulations Taq Plus used in RT-PCR amplified 1760bp lt; WP = 15 gt; single target gene. Clone sequencing and sequence analysis found that the Em Ⅱ / 3 and EM10 is different multi-room spines ball tapeworm elp gene group gene cDNA clone, constructed in this study the recombinant plasmid pEM10sc-11zf insert fragment containing the complete elp coding area, and EM10 and GenBank coding zone only one base type, i.e. nucleotide # 869 of G and A, respectively, the first 290 amino acids, respectively, after the translation is arginine (CGT), and histidine (CAT). Restriction enzyme digestion to the success of this study has elp gene coding sequence was subcloned in pET32a () and pQE30 (), multiple cloning sites to construct capable of expressing the target protein / thioredoxin (Trx) fusion protein in prokaryotic expression of recombinant the plasmid pETrx-ELP and prokaryotic non-fusion expression recombinant plasmid PQ-ELP. SDS-PAGE and Western blot analysis of two recombinant plasmids IPTG induction highly expressed the 83kDa TRX / ELP fusion protein and 67 kDa the ELP recombinant protein, respectively, accounting for 23% of the total bacterial protein and 14 to 17%. ELP size of 67 kDa recombinant protein by affinity chromatography, more than 90%. Restriction analysis confirmed Echinococcus elp gene coding region has been subcloned into the eukaryotic expression vector pcDNA3.1 (), was successfully constructed the eukaryotic expression recombinant plasmid PCD-ELP. RT-PCR, dot-ELISA and immunohistochemistry confirmed that the recombinant plasmid capable of expressing in mammalian cells COS7 multi-Echinococcus the ELP antigen (ie, the EM II / 3, EM10 antigen). Elp gene expression in E. coli Echinococcus non-fusion expression product of recombinant protein ELP as a subunit vaccine, alone or with Freund's adjuvant combined immune BALB / c mice after 2 weeks (A, B group), a large number of specific IgG1 antibodies are generated, group B also generates a small amount of specific IgG2b antibody. In addition to the B mice spleen PMNC the trace IFN-gamma, A group, the NS group IFN-gamma and group IL-4 and IL-12 were not detected. A, B group spleen lymphocyte proliferation slightly elevated lymphocyte CPM values ??were 2.8 and 10.8 times the NS group by Echinococcus tapeworm metacestode larvae the antigen (EMML CAG) or concanavalin stimulation, A, B group, especially group B lymphocyte proliferation. lt; WP = 16 GT; DNA vaccine immunization studies have shown that, the Ⅱ group 4 weeks after the first immunization can be detected in the serum to specific IgG1, IgG2a two antibodies, Ⅳ group can be detected by specific IgG1, IgG2a and IgG2b antibodies. OD value gradually increased with the increase in the number of immune and immune-specific antibodies. Six weeks after the first immunization group II and IV can be detected by specific antibodies of the IgG1, IgG2a and IgG2b three. The end of the experiment (ie after the first immunization 7w) species-specific antibody OD values ??were the highest. Ⅰ ~ Ⅳ group the mouse spleen PMNC culture supernatant IFN-gamma concentration, respectively 50,55,27.5 and GT; the 500pg/ml stimulated by specific antigen were 44,101.5,28.5 and GT; 500pg /

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