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Tissue-factor-based tumor peptide vaccine as well as preparation method and application thereof

A tumor polypeptide vaccine and tissue factor technology, which is applied in the field of tumor polypeptide vaccine preparation, can solve the problems of complex protein structure, difficult process, allergic reaction, etc.

Active Publication Date: 2014-01-22
JIANGSU HIGH WIT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they use the whole TF protein as the target and use antibodies or FVII for treatment, which has the disadvantages of complex protein structure, difficult process, and easy triggering of allergic reactions in the body.

Method used

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  • Tissue-factor-based tumor peptide vaccine as well as preparation method and application thereof
  • Tissue-factor-based tumor peptide vaccine as well as preparation method and application thereof
  • Tissue-factor-based tumor peptide vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: vector construction

[0026] 1.1 Cloning of AnsB-TTP-TF gene

[0027] The AnsB-TTP sequence comes from a plasmid vector already available in our laboratory. The AnsB-TTP-TF sequence can be obtained by adding the TF gene in two steps after the AnsB-TTP sequence by using the method of adding end PCR:

[0028] Design common upstream primers as:

[0029] A1: 5'-TAA TAC GAC TCA CTA TA - 3' (SEQ ID NO: 3)

[0030] Downstream primers are:

[0031] TF-down

[0032] The first step downstream primer TF-1: 5'- T GTC AGT AGT GTA GAA GCA TTT GGA TTT CCA GTC ACC GGT ACC AGA AGA GAT TAC AGA C-3' (SEQ ID NO: 4)

[0033] The second downstream primer TF-2: 5'- AAA AAGC TTA ACC GGT TTC GTC AGT CAA GTC GTA TTC AGT GTC AGT AGT GTA GAA GCA T-3' (SEQ ID NO: 5)

[0034] Upstream primers downstream contain restriction endonucleases Nco I site, the second downstream primer contains a restriction endonuclease Hind III site.

[0035] The upstream and downstream primers...

Embodiment 2

[0084] Embodiment two: transformation and screening

[0085] 2.1 Competent transformation

[0086] Take the above 5 μl connection solution to transform competent Escherichia coli BL21 (DE3) (for the preparation method, refer to the "Molecular Cloning Experiment Guide, Third Edition"), and the positive bacteria in the preliminary screening of kanamycin are then screened by enzyme activity assay, and the positive clones Extract the plasmid as a template, use the T7 promoter as the upstream primer, and use TF-2 as the downstream primer to express the vector pET28ansB-TTP-TF. The PCR conditions are the same as in Example 1.1; the clones that are positive by both screening methods are finally identified by DNA sequence assay to confirm the correct insertion of the AnsB-TTP gene.

[0087] Qualitative detection of asparaginase activity

[0088] The method for qualitative detection of asparaginase enzyme activity is the Naphthalene method: (boric acid buffer: H 2 BO 3 0.682...

Embodiment 3

[0090] Example 3: Expression and purification of fusion protein

[0091] 3.1 Engineering bacteria amplification of pET28ansB-TTP-TF

[0092] The positive engineering bacteria obtained after screening were activated respectively, and a ring of bacteria was taken, cultured with shaking at 30°C overnight, inoculated into fresh liquid LB medium containing kanamycin at 1% inoculum size, and cultured at 37°C. Cultivate for 3.5 hours after transfer, add 1% culture volume of 0.5 M lactose solution to induce, so that the final concentration of lactose in the culture solution reaches 5 mM, continue to culture at 28°C for 4 hours, centrifuge at 5000 rpm for 10 minutes, and collect the bacteria.

[0093] Lysis of bacteria

[0094] Lysis buffer: 10 mM phosphate buffer (PB, pH8.0), 1 mM EDTA. Add 5 ml of lysis buffer per gram of bacteria, add 80 μl of lysozyme (10 mg / ml) and 20 μl of DNAase I (1 mg / ml), and lyse at 37°C for 1 h. 4 0 C, 10000 rpm, 15 min, the supernatant and the pr...

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Abstract

The invention discloses a tissue-factor-based tumor peptide vaccine which is AnsB-TF fusion protein formed after a sequence fragment containing epitope targets in human TF genes is expressed. The invention also discloses a preparation method and application of the tumor peptide vaccine. According to the preparation method, a TF peptide fragment containing epitope targets is connected with an AnsB carrier through a genetic engineering means so as to obtain the tumor peptide vaccine taking tissue factors as target epitopes. The vaccine obtains prevention and treatment effects on tumors through active immunization on TF epitope targets. The active immunization not only can completely block the effect of TF on tumor surfaces, but also can be used for avoiding the trouble of multi-time administration.

Description

technical field [0001] The invention relates to a polypeptide vaccine, a preparation method and its application, especially a tumor polypeptide vaccine, a preparation method and its application. Background technique [0002] The coagulation function of tissue factor (TF) is well known. After it combines with activated coagulation factor VII (activated factor VII, F VIIa), it forms a complex of TFPF VIIa to initiate the extrinsic coagulation pathway; it can also activate coagulation factor XI, Activate the intrinsic coagulation system. It is one of the most active procoagulant substances in the body, and plays an important role in physiological hemostasis, coagulation process and various thromboembolic diseases. However, as the only transmembrane glycoprotein expressed on the cell surface in the blood coagulation system, whether TF has receptor properties and non-coagulation function has gradually become the focus of many scholars. Recent studies have shown that TF has the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P35/00C12N15/70
Inventor 刘文涛吴雪丰徐礼友杨政
Owner JIANGSU HIGH WIT BIOTECH CO LTD
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