Hepatitis C virus antigen polypeptide composition and hepatitis C virus vaccine
A technology of hepatitis C virus and antigen peptides, applied in the direction of virus antigen components, virus peptides, viruses, etc., can solve the problems of weak immunogenicity, unstable traits, and low specificity
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Embodiment 1
[0065] This embodiment is an in vitro synthesis of antigenic peptides, and the specific steps are as follows:
[0066] Entrusted Nanjing GenScript Company to artificially synthesize the five antigenic polypeptides from SEQ ID NO: 1 to SEQ ID NO: 5, the polypeptide sequence is as follows.
[0067] SEQ ID NO: 1 is ATRDGKLPTT;
[0068] SEQ ID NO: 2 is WGPISYANGSGLDE;
[0069] SEQ ID NO: 3 is ADTAACGDIING;
[0070] SEQ ID NO: 4 is AVQNEVTLT;
[0071] SEQ ID NO: 5 is SRGNHVSPTHYVPESD;
[0072] The five antigenic polypeptides were dissolved in dimethyl sulfoxide (DMSO) (product of Sigma Company) to a concentration of 10 mg / ml, sterilized by filtration with a 0.22 μM filter membrane, and stored at -80°C in aliquots.
Embodiment 2
[0074] This embodiment is the isolation and culture of DC cells, and the specific steps are as follows:
[0075] 1) Collect 80ml of peripheral blood from tumor patients, and obtain mononuclear cells by density gradient centrifugation.
[0076] 2) Dilute to 3-5×10 with RPMI1640 medium (Gibco company product) after counting 6 cells / ml, added to T75 culture flask for culture.
[0077] 3) At 37°C, 5% CO 2 After incubating in the incubator for 90 min, the non-adherent cells were collected by washing, which were T lymphocytes.
[0078] 4) Complete RPMI1640 medium containing 5% autologous serum, 1000 IU / ml recombinant human granulocyte macrophage colony-stimulating factor and 1000 IU / ml IL-4 (product of Sigma Company) was added to the adherent cells.
[0079] 5) The immature DC cells were collected on the fifth day, and the mature DC cells were cultured in complete RPMI1640 medium until collected on the eighth day.
[0080] Such as figure 1 As shown, the immature DCs were rounded ...
Embodiment 3
[0082] This example is the hepatitis C virus antigen polypeptide composition loaded DC cells and phenotype identification, the specific steps are as follows:
[0083] Collect 3×10 6 For the immature DC cells cultured to the fifth day in Example 2, use the 5 antigen polypeptides in Example 1 at 37°C and 5% CO 2 Load in the incubator for 2 hours, and the final concentration of each antigen polypeptide is 40 μg / ml. Divided into 9 groups:
[0084] ① DC cells loaded with the hepatitis C virus antigen polypeptide composition (HCV E1, HCV E2, HCV NS2, HCV NS3 and HCV NS4B antigen composition) of the present invention;
[0085] ② DC cells loaded with HCV E1, E2, NS2 and NS3 antigen polypeptide compositions;
[0086] ③ DC cells loaded with HCV E2, NS3 and NS4B antigen polypeptide compositions;
[0087] ④DC cells loaded with HCV E1 antigen polypeptide;
[0088] ⑤ DC cells loaded with HCV E2 antigen polypeptide;
[0089] ⑥DC cells loaded with HCV NS2 antigen polypeptide;
[0090] ...
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Abstract
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