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Phosphatidylinositol proteoglycan 3 nanometer antibody as well as preparation method and application thereof

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

Active Publication Date: 2020-10-02
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 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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  • Phosphatidylinositol proteoglycan 3 nanometer antibody as well as preparation method and application thereof
  • Phosphatidylinositol proteoglycan 3 nanometer antibody as well as preparation method and application thereof
  • Phosphatidylinositol proteoglycan 3 nanometer antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of GPC3 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, and the nucleotide sequence is shown in SEQ ID NO:10. (2) Use restriction endonucleases EcoRI and NotI (New England Biolabs Company) to double the GPC3ΔGPI-FLAG DNA fragment and the pcDNA3.1 vector (intermediate vector) containing SfiI restriction sites at both ends of the modified multiple cl...

Embodiment 2

[0039] Example 2 Screening of Nanobody Phage Library against GPC3

[0040] (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 in (A), the size of the purified protein is between 72-85kD, and the band is single, indicating high purity. figure 2 (B) Western blot detection results using the FLAG tag antibody of the GPC3ΔGPI-FLAG recombinant protein. The results show 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 caused by GPC3 glycosylation. The arrow indicates the target band position of the GPC3ΔGPI-...

Embodiment 3

[0041] Preparation of Example 3 Nanobodies

[0042] 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 G64 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 amino acid sequences of CDR1, CDR2, and CDR3 of the three complementary regions are shown in SEQ ID No:1, SEQ ID No:2, and SEQ ID No:3 respectively, and the nucleotide sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID As shown in NO:4, SEQ ID NO:5, and SEQ ID NO:6, the amino acid sequen...

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Abstract

The present invention discloses a phosphatidylinositol proteoglycan 3 (Glypican-3, GPC3) nanometer antibody and a preparation method thereof. The nanometer antibody has three complementarity determining regions: CDR1, CDR2 and CDR3, wherein the CDR1 comprises an amino acid sequence as shown in SEQ ID NO.1, and the CDR2 comprises an amino acid sequence as shown in SEQ ID NO.2, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO.3. The nanometer antibody disclosed by the invention can be used for specifically identifying liver cancer cells with high GPC3 expression by combining with GPC3 expressed by a cell membrane. The antibody has high stability in serum, can be used for function research of the GPC3, and can also be used for development of diagnosis reagents and treatmentdrugs of the hepatocellular carcinoma.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a nanobody of Glypican 3 and its preparation method and application. 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 significance to improve the survival of liver cancer patients. Targeted drugs have become one of the most...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N1/21G01N33/68G01N33/574A61K39/395A61K47/68A61P35/00C12R1/19
CPCC07K16/303C12N15/70G01N33/68G01N33/57438G01N33/57492A61K47/68A61P35/00C07K2317/569C07K2317/565C07K2317/567C07K2317/94A61K2039/505
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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