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A kind of nanobody of Glypican 3 with outstanding thermal stability and preparation method thereof

A phosphatidylinositol and nano-antibody technology, applied in the biological field, can solve the problems of low antibody stability, complicated preparation process, and high cost, and achieve the effects of high antibody stability, simple operation, and low cost

Active Publication Date: 2022-02-08
ZHUHAI INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been reports of monoclonal antibodies that specifically recognize GPC3. However, in view of the limitations of traditional antibodies such as low stability, complicated preparation process, and high cost, there is an urgent need to develop new antibodies against GPC3.

Method used

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  • A kind of nanobody of Glypican 3 with outstanding thermal stability and preparation method thereof
  • A kind of nanobody of Glypican 3 with outstanding thermal stability and preparation method thereof
  • A kind of nanobody of Glypican 3 with outstanding thermal stability and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preparation of embodiment 1GPC3 recombinant protein

[0037] Prepare GPC3 recombinant protein with FLAG tag at the carboxy-terminus.

[0038] (1) According to the coding gene sequence of GPC3 in NCBI (NCBI Reference Sequence: NM_004484.4), and based on the amino acid sequence of GPC3 in UniProt (UniProt Reference Sequence: P51654-1), the F359S mutation was performed, and the GPI region ( 1690-1740bp), the fusion sequence of GPC3ΔGPI and FLAG tag missing the GPI site was artificially synthesized (Shenzhen Huada Gene Technology Co., Ltd.), with EcoRI and NotI restriction sites at the 5' end and 3' end, respectively. The amino acid sequence encoded by GPC3ΔGPI is shown in SEQ ID NO:9

[0039]

[0040]

[0041]

[0042] Its nucleotide sequence is shown in SEQ ID NO:10

[0043]

[0044]

[0045]

[0046] (2) Use restriction endonucleases EcoRI and NotI (New England Biolabs Company) to double the GPC3ΔGPI-FLAG DNA fragment and the pcDNA3.1 vector (inte...

Embodiment 2

[0048] Example 2 Screening of Nanobody Phage Library against GPC3

[0049] (1) Coating antigen: According to the instructions of FLAG M2 magnetic beads, the GPC3ΔGPI-FLAG protein secreted into the culture supernatant was purified to prepare GPC3ΔGPI-FLAG protein-coated magnetic beads, which were stained with Coomassie brilliant blue and immunoblotted. Test for verification. like figure 2 As shown, from the results of Coomassie Brilliant Blue staining in the left figure, it can be seen that the purified protein is between 72-85kD in size and has a single band, indicating a high purity. The image on the right shows the results of Western blot detection using the FLAG tag antibody of the GPC3ΔGPI-FLAG recombinant protein, showing that the protein size is consistent with the Coomassie brilliant blue staining results, indicating that the purified protein is the target protein of GPC3ΔGPI-FLAG. GPC3 protein has several glycosylation sites, and the trailing diffuse bands should be...

Embodiment 3

[0050] Preparation of Example 3 Nanobodies

[0051] In Example 2, after completing the third round of screening for phage infection, Escherichia coli SS320 was coated on a plate, and single clones containing phage plasmids were picked for sequencing. The gene sequences of each antibody clone were analyzed and compared using Vector NTI software, and the clones with the same sequence of complementary regions CDR1, CDR2, and CDR3 were regarded as the same clone. According to the sequencing results, one of the clones with a high repetition rate was selected and labeled as G8 clone. The DNA sequence shown is shown in SEQ ID NO:8, and the encoded amino acid sequence is shown in SEQ ID NO:7. The nucleotide fragments of the selected G8 nanobody were connected to the expression vector pET22b by PCR amplification, restriction endonuclease digestion and T4 ligase connection. PCR primers for increasing the nucleotide fragment of the Nanobody: upstream primer catgactagt caagttcaat tagtc (...

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Abstract

The present invention relates to a nanobody of Glypican 3 with outstanding thermal stability and a preparation method thereof, and also relates to the amino acid sequence of the nanobody, a gene coding sequence and an expression vector capable of expressing the nanobody and host cell. The amino acid sequence of the nanobody to GPC3 provided by the present invention is shown in SEQ ID NO:7, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:8. The GPC3 nanobody involved in the present invention can specifically recognize liver cancer cells with high GPC3 expression by binding to the GPC3 expressed on the cell membrane. The antibody has outstanding thermal stability, and can be used in the functional research of GPC3, and can also be used in the development of diagnostic reagents and therapeutic drugs for hepatocellular carcinoma.

Description

technical field [0001] The invention relates to a nanobody of Glypican 3 with outstanding thermal stability and a preparation method thereof, and also relates to a gene encoding the same, an expression vector, a host cell and related applications, belonging to the field of biotechnology. Background technique [0002] Liver cancer is known as the "King of Cancer" and is the third cancer with the highest mortality rate. The number of new cases and deaths of liver cancer in China accounts for more than half of the global total. Hepatocellular carcinoma (HCC) is the most important form of liver cancer, accounting for about 75%. Due to the hidden early symptoms of liver cancer and the limitation of diagnostic methods, most patients with liver cancer are already in the advanced stage of liver cancer when they are diagnosed, and the prognosis of advanced liver cancer is poor, so the treatment is very difficult. Therefore, the early diagnosis of liver cancer is of great significan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N1/21G01N33/574G01N33/68A61K47/68A61K45/00A61K35/17A61P35/00C12R1/19
CPCC07K16/303C07K16/005C12N15/70G01N33/57438G01N33/57492G01N33/6857A61K47/6801A61K45/00A61K35/17A61P35/00C07K2317/569C07K2317/565C07K2317/567C07K2317/94C07K2317/14
Inventor 王文义徐畅姜长安
Owner ZHUHAI INST OF ADVANCED TECH
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