Preliminary Study On Subunit Vaccine And Nucleic Acid Vaccine Of Echinococcus Multilocularis

ObjectiveTo construct prokaryotic expression vector of Echinococcus multilocularis elp gene, express in Escherichia coli and purify the recombinant protein; To construct eukaryotic expression vector of elp gene, determine the expression ability of the vector by temporary expression in a mammalian cell COS7 and purify the eukaryotic expression vector; To observe the immune responses of BALB/c mice induced by the subunit vaccine(the recombinant protein of ELP expressed in E. coli) and by the nucleic acid vaccine(the eukaryotic expression vector of E. multilocularis elp gene); To compare the immunization effect of the two vaccines and examine the adjuvant actions of Freund's adjuvant for the subunit vaccine and the eukaryotic expression vector of mouse IL-12 for the nucleic acid vaccine of E. multilocularis.MethodsE. multilocularis metacestode larva(EMML) was prepared from artificially-infected gerbils. EMML total RNA was extracted with a nucleic acid extraction kit. PCR primers containing cutting sites of restriction-endonuclases were designed based on the cDNA sequence of E. multilocularis elp gene downloaded from GenBank. The objective gene was obtained with a RT-PCR technique from the total RNA of EMML. The effects of 5 commercialized nucleic acid extraction kits and different DNA polymerases for amplification of DNA long fragments with RT-PCR were observed and compared at the same time. The PCR product was inserted into the multiple cloning site of pGEM-11zf with a cloning technique. Then the insertion was sequenced and analyzed.The objective DNA fragment containing the full length of elp gene coding sequence was subcloned into prokaryotic expression vectors pET32a(+) and pQE30(+). Expression of elp gene in E. coli was induced with IPTG and the recombinant protein was identified with Western blot.Eukaryotic expression vector of elp gene was constructed by subcloning the objective DNA fragment into pcDNA3.1(+). And then the recombinant eukaryotic expression vector was transfected into mammalian cells COS7. The expression ability of the recombinant plasmid (pcD-ELP) was confirmed by detection of transcription product of elp gene in the cells with RT-PCR,Dot-ELISA and immunohistochemistry were used to examine the expressed protein coded by elp gene in COS7 cells. Large quantity of the recombinant protein ELP expressed in E. coli was purified with an affinity chromatography column and the recombinant eukaryotic expression vector of elp gene was purified with an endotoxin-free kit. Seventy-two BALB/c mice aged 4-6 weeks were divided into 7 groups, 10 in each of the first 6 groups and 12 in the last group. Males and females in each group were the same in number. Group A: per capita recombinant protein ELP 100μg; group B: per capita recombinant protein ELP 100μg mixed with equal volume of Freund's adjuvant(complete Freund's adjuvant for the first dose and incomplete Freund's adjuvant for the second and third doses); group NS: normal saline as control; group Ⅰ: per capita blank plasmid pcDNA3.1(+) 100μg and 0.75% bupivacaine 480μl; group Ⅱ: per capita recombinant plasmid pcD-ELP 100μg and 0.75% bupivacaine 480μl; group Ⅲ: mouse interleukine-12 eukaryotic expression plasmid(pIL-12) 100μg and 0.75% bupivacaine 480μl; group Ⅳ: pcD-ELP 100μg, pIL-12 100μg and 0.75% bupivacaine 480μl. Subcutaneous injection at multiple sites was adopted for group A and group B. For group NS and groups Ⅰ to Ⅳ, the volume of solution was adjusted to 140μl with NS and intramuscularly injected bilaterally at quadriceps femoris. Three times of immunization were given to all groups at a two-week interval. Serum antibodies of IgG1, IgG2a and IgG2b against the recombinant protein ELP were detected by ELISA at the time of pre-immunization (week 0) and at the ends of week 2, 4, 6 and 7 after the first immununization. At the end of week 7, Sandwich ELISA kits were used to examine interferon-γ(IFN-γ), interleukin-4(IL-4) and interleukin-12(IL-12) produced by peripheral monon...