Ran Xu-hua, Wen Xiao-bo, Wang Chun-ren, Li Xiao-juan, Wei Xiao-man
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2014 Aug;32(4):289-92.
To analyze the immunoreactivity of recombinant cathepsin L-like proteases (CatL) protein of Fasciola hepatica and its immunogenicity in SD rats.
The E. coli BL21(DE3) cells harbouring recombinant plasmid pET30a-FhCatL were inoculated in LB medium, and the protein expression was induced with IPTG. The recombinant protein FhCatL was analyzed by SDS-PAGE and the immunoreactivity was identified by Western blotting with sera from Fasciola hepatica-infected goat as the primary antibody. Twenty SD rats were randomly divided into immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhCatL protein. All the rats received three immunizations at 3-week intervals. The adjuvant control group with 10 SD rats received only adjuvants emulsified with the same amount of PBS. Serum samples were collected at the day before the second and final immunization, 3, 6, and 9 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by methyl thiazolyl tetrazolium (MTT).
The molecular weight of purified FhCatL was about Mr 42,000. The recombinant FhCatL was recognized by pool sera of goats naturally infected with F. hepatica. The titer of specific antibody IgG in SD rats induced by the recombinant protein against CatL protein was significantly higher than that of the control, and the antibody titer reached the peak at three weeks after the final immunization (1 : 102,400). The stimulation index of splenocytes in immunized group was 2.176 +/- 0.047, which was significantly higher than that of the control (1.171 +/- 0.032) (P<0.05).
The recombinant FhCatL protein bears stronger immnoreactivity and immunogenicity.
