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TK1 antibody, kit and application of TK1 antibody

A kit and antibody technology, applied in the field of immunology, can solve the problems of high resistance of liver cancer to chemotherapy and difficulty in prolonging life, and achieve the effect of reducing the rate, increasing the frequency of apoptosis and high affinity

Active Publication Date: 2021-04-13
SHENZHEN HUARUI TONGKANG BIOTECHNOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Further, since liver cancer is highly resistant to chemotherapy, it is difficult even to temporarily stabilize the tumor through treatment, that is, prolong life without causing tumor proliferation.

Method used

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  • TK1 antibody, kit and application of TK1 antibody
  • TK1 antibody, kit and application of TK1 antibody
  • TK1 antibody, kit and application of TK1 antibody

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0038] Step 1: Preparation of TK1 antigen

[0039] Inoculation: Synthesize the full-length TK1 gene sequence into the vector pET21a-TK1-C6His, inoculate pET21a-TK1 BL21 expression bacteria into 5ml ampicillin-resistant LB medium from a -80°C refrigerator, and culture overnight at 37°C 220rpm (do not melt the expression bacteria during inoculation ).

[0040] Large-scale induction: Inoculate BL21(DE3) bacterial solution in 1000ml ampicillin-resistant TB medium at a ratio of 1:200 the next day. Cultivate at 37°C and 220rpm. When the bacteria enter the logarithmic growth phase (OD is about 0.6, the cultured bacteria can hardly be seen through, and in the state of cloud and fog), cool the culture bottle in the ice-water mixture, add IPTG with a final concentration of 1mM, and incubate at 25°C. °C induction for 15 hours.

[0041] Collect the expression bacteria: centrifuge the bacterial solution at 6000rpm for 15min to remove the supernatant, resuspend the bacteria (200ml / L expre...

Embodiment 1

[0114] Step 1: After the hepatic tumor cell lines Hep G2, Hep 3B and the 0.1 μg / ml monoclonal antibody containing the SSTK mouse hybridoma cell line 43# of the present invention are fully mixed (2 × 10 4 ), sterilized by filtration in a biosafety cabinet with a 0.22um sterile filter head (Cat. No.: SLGP033RB), in MEM Alpha basic cells containing 15% fetal bovine serum (ExCell Bio, Cat. No. FS500) and without antibiotics or other reagents Culture medium (GIBCO, catalog C12571500BT) for 72h;

[0115] Step 2: Sterilize the liver tumor cell lines Hep G2 and Hep 3B0.22um by filtration, and then culture them in MEM Alpha basic cells containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) without antibiotics or other reagents Base (GIBCO, catalog C12571500BT) for 72 hours;

[0116] Step 3: Perform cell viability test (Cell Counting Kit-8, MedChemExpress)

[0117] Viability calculation: tumor cell viability (%)=(absorbance of experimental well-absorbance of blank medium...

Embodiment 2

[0119] Step 1: After the hepatic tumor cell lines Hep G2, Hep 3B and the 1 μg / ml monoclonal antibody containing the SSTK mouse hybridoma cell line 43# of the present invention are fully mixed (2 × 10 4 ), cultivated for 72h in the MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) and without antibiotics or other reagents;

[0120] Step 2: Culture the liver tumor cell lines Hep G2 and Hep 3B in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) without antibiotics or other reagents 72h;

[0121] Step 3: Carry out cell viability test (Cell Counting Kit-8, MedChemExpress) Viability calculation: tumor cell viability (%)=(absorbance of experimental well-absorbance of blank medium control) / (absorbance of untreated control well- Blank medium control absorbance) × 100%

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Abstract

The invention discloses a TK1 antibody, a kit and application of the TK1 antibody. The TK1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 with an amino acid sequence shown as SEQ ID NO: 1, a heavy chain CDR2 with an amino acid sequence shown as SEQ ID NO: 2 and a heavy chain CDR3 with an amino acid sequence shown as SEQ ID NO: 3; and the light chain variable region comprises a light chain CDR1 with an amino acid sequence shown as SEQ ID NO: 4, a light chain CDR2 with an amino acid sequence shown as SEQ ID NO: 5, and a light chain CDR3 with an amino acid sequence shown as SEQ ID NO: 6. The hepatocellular tumor specific antibody prepared by the invention specifically binds to hepatocellular tumor cells, does not block the normal survival activity of normal cells, has high affinity and stable expression, and can significantly reduce the proliferation rate of human hepatocellular tumor cells and increase the cell apoptosis frequency. The antibody can be used for diagnosis or treatment, and is a novel effective targeted therapeutic drug for blocking tumor cell proliferation, especially liver cancer.

Description

technical field [0001] The present invention relates to the field of immunology, in particular to a TK1 antibody, kit and application thereof. Background technique [0002] Liver cancer is one of the common malignant tumors in my country, with more than 500,000 new cases every year, and almost the same number of deaths due to low treatment efficiency. Hepatocellular carcinoma accounts for approximately 80% of all liver cancers and is rarely curable. Its 5-year survival rate is only about 10%, and the survival time after diagnosis is usually less than 6 months. [0003] Regarding the treatment of liver cancer, there are surgical hepatectomy targeting visible lesions, radio wave ablation therapy, ethanol injection therapy, and microwave coagulation necrosis therapy, etc., but these local therapies may not be suitable. [0004] At present, for advanced cancer patients who cannot adapt to the above-mentioned therapy and hepatic artery embolization surgery, or patients who rela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13A61K39/395A61P35/00
CPCC07K16/40A61P35/00C07K2317/56C07K2317/565C07K2317/73A61K2039/505
Inventor 李劲张波李惠军周际艾伦·何斯文·斯库格
Owner SHENZHEN HUARUI TONGKANG BIOTECHNOLOGICAL
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