title: [Construction of eukaryotic recombinant plasmid of periodic Brugia malayi CPI gene and its immunity].
abstract: OBJECTIVE
  To observe the immune responses elicited in BALB/c mice by DNA vaccine encoding cysteine protease inhibitor (CPI) of periodic Brugia malayi cloned in vector pcDNA3.1.
  
  
  METHODS
  Specific primers were designed on the basis of known sequences of CPI gene from periodic B. malayi. The desired gene fragment was amplified by PCR from cDNA, inserted into cloning vector, pGEM-T, and sub-cloned into pcDNA3.1 to construct pcDNA3.1-BmCPI Forty-eight mice were randomly divided into 4 groups, i.e. normal control group, pcDNA3.1(+) group, pcDNA3.1-BmCPI group, and pcDNA3.1-BmCPI/CpG group injected with PBS 100 microl, pcDNA3.1 100 microg, pcDNA3.1-BmCPI 100 microg and pcDNA3.1-BmCPI 100 microg+CpG 30 microg, respectively on left hind leg of each mouse. All mice received three immunizations with 2-week interval. At the 4th week after the last immunization the muscle around injection spot was collected, in which the level of BmCPI mRNA was detected by RT-PCR. The stimulation index (SI) of spleen lymphocytes was measured by MTI method and the levels o f secreted IL-4 and IFN-gamma in serum were detected by ELISA.
  
  
  RESULTS
  The recombinant plasmid pcDNA3.1-BmCPI was constructed and the length of the gene fragment was 621 bp. The results showed that BmCPI gene in the muscle of the immunized mice was detected by PCR. At the 4th and 6th weeks after immunization, the SI of the two immunized groups was significantly higher than normal control group and pcDNA3.1(+) group (53.789 +/- 1.937, 59.735 +/- 4.139, and 61.975 +/- 1.029) (P < 0.05). No significant difference existed between pcDNA3.1BmCPI group and pcDNA3.1-BmCPI/CpG group (P > 0.05). Serum IFN-gamma in pcDNA3.1-BmCPI group and pcDNA3.1-BmCPI/ CpG group increased from the 2nd to the 6th week after the last immunization with the value of 69.544 +/- 3.145 and 106.069 +/- 7.518, 120.019 +/- 5.968 and 136.229 +/- 7.198, 149.109 +/- 2.700 and 178.429 +/- 1.126, respectively. The levels of IFN-gamma in serum from the immunized mice were significantly higher than those of normal control group and pcDNA3.1(+) group (28.264 +/- 1.129, 35.179 +/- 1.029, and 40.110 +/- 1.176, respectively) (P < 0.05). There was a significant difference between the two immunized groups at the 2nd and the 6th weeks after the last immunization (P < 0.05). The level of IL-4 in serum from the immunized mice was significantly higher than those of normal control group and pcDNA3.1(+) group at the 4th and the 6th weeks after the last immunization (P < 0.05). No significant difference was noted in IL-4 level between pcDNA3.1-BmCPI group and pcDNA3.1-BmCPI/CpG group (P > 0.05).
  
  
  CONCLUSIONS
  The recombinant eukaryotic plasmid pcDNA3.1-BmCPI was transcribed in vivo and elicited immune responses in mice.
authors.forename: Sai-Nan
authors.initials: SN
authors.lastname: Zhang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Fang
authors.forename: Shi-Juan
authors.initials: SJ
authors.lastname: Lu
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Bang-Sheng
authors.initials: BS
authors.lastname: Xu
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000235:genetics* / Q000276:immunology*; D015853:Cysteine Proteinase Inhibitors / Q000276:immunology*; D015870:Gene Expression; D005849:Gerbillinae; D007111:Immunity, Cellular; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D019444:Vaccines, DNA / Q000276:immunology*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D019444:Vaccines, DNA