CETP antigenic peptide and fusion protein and their composition and applications
A fusion protein and antigenic peptide technology, applied in the field of antigenic peptides, can solve the problems of immunogenicity and insufficient efficiency
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[0109] Materials and Method
[0110] A DNA fragment of 6 repeated sequences encoding the human CETP epitope was constructed, and then it was fused with rabbit Fc and the fusion protein was expressed in E. coli BL21 (DE3).
[0111] As previously described (Hsu et al. 2000, Cancer Res. 60:3701-3705), the peptide PEHLLVDFLQSL encoding the human CETP epitope was generated by template repeat polymerase chain reaction (TR-PCR) (Gaofu et al. 2004, Vaccine 22: 3187-3194) DNA fragments of 6 repeating sequences ( figure 1 ). The adapter primers (Table 1) were then used to subject the TR-PCR product to adapter PCR to form restriction sites at the 5'end and 3'end to facilitate further subcloning. The 200-300bp linker PCR product ( figure 1 B) Eluted and cloned into T-Easy vector (Promega). The clones containing 6 copies of the CETP epitope were identified by sequencing and subcloning at the 3'end of the region encoding the Fc domain of rabbit IgG into a modified plasmid pET21b vector (Novage...
example 1
[0135] Example 1 Production of Fc-CETP6 vaccine
[0136] Generate DNA encoding 6 repeats of CETP epitope and induce and express the fusion protein in E. coli strain BL21 (DE3), such as figure 1 Shown in. The fusion protein Fc-CETP6 was purified by affinity chromatography on a histidine (His) binding column. The purity of the Fc-CETP6 fusion protein was checked by Coomassie Blue staining and Western blotting, and the purity reached 95%. The Fc-CETP purified by histidine binding 6 Purity: Lanes 1 to 3 show the staining results of Coomassie Blue for flow-through, washing and elutes, respectively, while Lane 4 shows that the eluate uses anti-rabbit IgG (Fc) Western blot analysis ( figure 1 C).
example 2
[0137] Example 2. In rabbits fed with HFC feed, Fc-CETP 6 The vaccine raises antibodies against CETP and reduces CETP activity
[0138] In order to confirm that the anti-CETP antibody is produced and the antibody reacts with CETP in the circulation, plasma anti-CETP titer and plasma CETP activity are measured. figure 2 A shows that in Fc-CETP 6 Anti-CETP antibodies were detected in the group from week 12 and the titer increased until the end of the study at week 24. figure 2 B shows that in the two groups fed with HFC feed, plasma CETP activity increased in a time-dependent manner, but in Fc-CETP 6 In the group, the plasma CETP activity was significantly lower. These results show that the injection of Fc-CETP 6 The production of antibodies against CETP is induced, which then reduces CETP activity.
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