关键词: 马来丝虫, 半胱氨酸蛋白酶抑制剂, 真核表达载体, 免疫

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 μl， pcDNA3.1 100 μg， pcDNA3.1-BmCPI 100 μg and pcDNA3.1-BmCPI 100 μg+CpG 30 μg， 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 MTT method and the levels of secreted IL-4 and IFN-γ 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 pcDNA3.1-BmCPI group and pcDNA3.1-BmCPI/CpG group (P>0.05). Serum IFN-γ 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-γ 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）.   Conclusion   The recombinant eukaryotic plasmid pcDNA3.1-BmCPI was transcripted in vivo and elicited immune responses in mice.

Key words: Brugia malayi, Cysteine protease inhibitor, Eukaryotic recombinant plasmid, Immunity