Nanobody of anti-GPC 3 (Glypican 3) protein and preparation method and application of nanobody

A nanobody and protein technology, applied in the biological field, can solve problems such as the preparation method of nanobodies without anti-GPC3 protein

Active Publication Date: 2018-10-16
XINJIANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no nanobody and efficient preparation method against GPC3 protein

Method used

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  • Nanobody of anti-GPC 3 (Glypican 3) protein and preparation method and application of nanobody
  • Nanobody of anti-GPC 3 (Glypican 3) protein and preparation method and application of nanobody
  • Nanobody of anti-GPC 3 (Glypican 3) protein and preparation method and application of nanobody

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Experimental program
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Effect test

Embodiment 1

[0049] Prokaryotic expression of embodiment 1 human GPC3 gene

[0050] 1. Construction of prokaryotic expression cells

[0051] According to the nucleotide sequence design of the human GPC3 cDNA provided by the GenBank database, a pair of primers for amplifying the full-length signal peptide of the GPC3 gene, the sequence of the primers is as follows:

[0052] Primer1 (SEQ ID NO:3):

[0053] 5'-CGGAATTCATGCAGCCCCCGCCGCCGCCGCCGGACGCCACCTG-3

[0054] Primer2 (SEQ ID NO:4):

[0055] 5'-CCCTCGAGTCAGTGCACCAGGAAGAAGAAGCAC-3'

[0056] The upstream and downstream respectively have EcoR I and Xho I restriction sites, use the above primers to carry out PCR amplification, use 1% agarose gel electrophoresis to detect the size of the PCR fragment, use the PCR product purification and recovery kit to recover the PCR product, and the recovered After double digestion of the target fragment and pET28a plasmid at 37°C overnight, the double digested target fragment and pET28a vector were rec...

Embodiment 2

[0063] Example 2 Construction and screening of Xinjiang Bactrian camel phage display library

[0064] 1. Fusion protein His-GPC3 to immunize Xinjiang Bactrian camels

[0065] Mix 10 mg of purified His-GPC3 protein with an equal volume of Freund's adjuvant, immunize Xinjiang Bactrian camels (subcutaneous injection at 3-5 points in the neck), boost once every 2 weeks, and immunize 5 times in total (first use Complete Freund's adjuvant, the rest with incomplete Freund's adjuvant). Before each immunization and 14 days after the final immunization, blood was collected from the jugular vein, and the serum was separated for titer detection. It was found that the expected immune effect was achieved and the immune response was stimulated ( Figure 6 ). 14 days after the final immunization, the spleen tissue of camels with immune response was collected.

[0066] 2. RNA Extraction from Camel Spleen Tissue

[0067] Take 0.5g of spleen tissue sample and place it in a pre-cooled mortar,...

Embodiment 3

[0108] Example 3pET28a-VHH GPC3 Construction of prokaryotic expression vector

[0109] 1. pET28a-VHH GPC3 Construction of prokaryotic expression vector

[0110] With reference to embodiment 1 method, will code VHH GPC3 The gene was connected to the prokaryotic expression vector pET28a plasmid.

[0111] 2. VHH GPC3 Prokaryotic Expression of Nanobodies

[0112] The identified correct pET28a-VHH GPC3 The prokaryotic plasmid was transformed into Escherichia coli BL21 expression strain, which was soluble expression. After purification by affinity chromatography, SDS-PAGE electrophoresis and Western Blot identified its molecular weight as 13kD ( Figure 10 ).

[0113] 3. VHH GPC3 Determination of Nanobody Affinity

[0114] 3.1 Sandwich ELISA method

[0115] Antigen coating: Dilute the purchased unlabeled eukaryotic protein GPC3 with carbonate coating buffer to a concentration of 2 μg / mL, add 100 μL to each well in a 96-well plate, and coat the ELISA plate with 10 μg / mL BSA...

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Abstract

The invention discloses a nanobody of the anti-GPC 3 (Glypican 3) protein and a preparation method and application of the nanobody. A prokaryotic expression GPC 3 fusion protein is used to immunize acamel, the RNA in a spleen tissue of the camel is extracted and is used as a template to implement inverse transcription to form cDNA, the cDNA is constructed to a carrier of an outer capsid protein of a bacteriophage to construct a bacteriophage display library, and the storage capacity and the titer are calculated. Then the nanobody specifically combined with the GPC 3 is screened from the library according to the antigen-antibody affinity principle after elutriating for three times, a gene of the specific antibody is then obtained, an expression carrier is constructed, the prokaryotic expression is implemented, the expression carrier is purified and identified, and the required nanobody is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nanobody against GPC3 protein, and also relates to a preparation method and application of the nanobody. Background technique [0002] Liver cancer is one of the most common malignant tumors in the world, and its morbidity and mortality are increasing year by year, seriously threatening human health. The treatment of liver cancer mainly includes liver transplantation, tumor resection and non-resective local therapy such as hepatic arterial chemoembolization. Since liver cancer, especially primary liver cancer (HCC), is prone to metastasis in the early stage and recurrence after treatment, it is of great significance to find an indicator that can accurately judge prognosis and an effective target for liver cancer treatment. [0003] Early diagnosis and treatment is one of the key factors to improve the curative effect of primary hepatocellular carcinoma. Glypican 3 (Glypican 3, GP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/10A61K39/395A61P35/00G01N33/574
CPCA61P35/00C07K16/005C07K16/303C07K2317/22C07K2317/569C07K2317/76C12N15/1037
Inventor 夏丽洁腾桥张富春李金耀
Owner XINJIANG UNIVERSITY
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