Phosphatidylinositol proteoglycan 3 nano antibody with outstanding thermal stability and preparation method thereof

A phosphatidylinositol and nanobody technology, applied in the biological field, can solve problems such as high cost, low antibody stability, and complicated preparation process

Active Publication Date: 2020-10-30
珠海中科先进技术研究院有限公司
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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

Method used

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  • Phosphatidylinositol proteoglycan 3 nano antibody with outstanding thermal stability and preparation method thereof
  • Phosphatidylinositol proteoglycan 3 nano antibody with outstanding thermal stability and preparation method thereof
  • Phosphatidylinositol proteoglycan 3 nano antibody with outstanding thermal stability and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of embodiment 1GPC3 recombinant protein

[0033] Prepare GPC3 recombinant protein with FLAG tag at the carboxy-terminus.

[0034] (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 Its nucleotide sequence is shown in SEQ ID NO:10 (2) Use restriction endonucleases EcoRI and NotI (NewEngland Biolabs) to double-enzyme the GPC3ΔGPI-FLAG DNA fragment and the pcDNA3.1 vector (intermediate vector) containing SfiI restriction sites at both ends of the modified mu...

Embodiment 2

[0036] Example 2 Screening of Nanobody Phage Library against GPC3

[0037] (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. Such as 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...

Embodiment 3

[0038] Preparation of Example 3 Nanobodies

[0039] 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 invention relates to a phosphatidylinositol proteoglycan 3 nano antibody with outstanding thermal stability and a preparation method thereof, and also relates to an amino acid sequence and a genecoding sequence of the nano-antibody, and an expression vector and a host cell capable of expressing the nano-antibody. The amino acid sequence of the GPC3 nano antibody provided by the invention is as shown in SEQ ID NO 7, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO 8. The GPC3 nano antibody can be combined with GPC3 expressed by a cell membrane to specifically recognize hepatoma carcinoma cells highly expressed by GPC3. The antibody has outstanding thermal stability, can be used for functional research of GPC3, and can also be used for development of diagnosticreagents 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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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 珠海中科先进技术研究院有限公司
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