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Nano-antibody of glypican 3 with outstanding high stability and preparation method of nano-antibody

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: 2020-11-10
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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  • Nano-antibody of glypican 3 with outstanding high stability and preparation method of nano-antibody
  • Nano-antibody of glypican 3 with outstanding high stability and preparation method of nano-antibody
  • Nano-antibody of glypican 3 with outstanding high stability and preparation method of nano-antibody

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Experimental program
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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 the amino acid sequence of GPC3 in Uni Prot (Uni Prot Reference Sequence: P51654-1), the F359S mutation was performed to remove its GPI Region (1690-1740bp), artificially synthesized GPC3ΔGPI and FLAG tag fusion sequence without GPI site (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 (Met Ala Gly Thr ValArg Thr Ala Cys Leu Val Val Ala Met Leu Leu Ser Leu Asp Phe Pro Gly Gln AlaGln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser Phe Phe GlnArg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp LeuGln Val Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Lys...

Embodiment 2

[0035] Example 2 Screening of Nanobody Phage Library against GPC3

[0036] (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

[0037] Preparation of Example 3 Nanobodies

[0038] 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 G11 clone, the DNA sequence shown was shown in SEQ ID NO:8, and the encoded amino acid sequence was shown in SEQ ID NO:7. The nucleotide fragments of the selected G11 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 tag...

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Abstract

The invention relates to a nano-antibody of glypican 3 with outstanding high stability and a preparation method of the nano-antibody, and also relates to an amino acid sequence of the nano-antibody, agene coding sequence, an expression vector capable of expressing the nano-antibody and a host cell. The amino acid sequence of the nano-antibody of GPC3 is shown as SEQ ID NO: 7, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 8. The nano-antibody of the GPC3 can specifically identify hepatoma carcinoma cells with high GPC3 expression by binding to the GPC3 expressed by cellmembranes. The antibody has outstanding high stability, can be used for the functional study of the GPC3 and also can be used for the development of the diagnostic reagents and therapeutic drugs of the hepatocellular carcinoma.

Description

technical field [0001] The invention relates to a nanobody of Glypican 3 with outstanding high 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 significance ...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N1/21G01N33/68G01N33/574A61K47/68A61P35/00C12R1/19
CPCC07K16/303C07K16/005C12N15/70G01N33/6857G01N33/57438G01N33/57492A61K47/68A61P35/00C07K2317/569C07K2317/565C07K2317/567C07K2317/94
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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