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Human monoclonal antibody against protein tag streptactin

A technology for cloning antibodies and tags, applied in immunoglobulins, antibacterial immunoglobulins, peptides, etc., which can solve the problems of poor specificity and achieve the effects of good specificity, enhanced anti-aggregation, and high affinity

Active Publication Date: 2021-09-03
武汉班科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing antibodies against this sequence are all animal-derived, but there are problems such as poor specificity in detection. Here, we use the human Fab phage display library to screen the human monoclonal antibody against this tag. It can be used for the purification and detection of proteins containing this tag, improving the specificity of detection, etc.

Method used

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  • Human monoclonal antibody against protein tag streptactin
  • Human monoclonal antibody against protein tag streptactin
  • Human monoclonal antibody against protein tag streptactin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Obtaining of A1 antibody sequence

[0014] According to the existing literature (Zhu Z, Dimitrov DS, Methods Mol Biol, 2009), a human Fab phage display library was constructed, and based on the library, the protein containing the Streptactin tag was screened for three rounds, and the monoclonal phage ELISA , sent the positive clones for sequencing, obtained the base sequence of the A1 candidate clone, and obtained its amino acid sequence through the amino acid translation software (https: / / web.expasy.org / translate / ), the light chain amino acid sequence is as follows As shown in SEQ No.1, its heavy chain amino acid sequence is shown in SEQ No.2.

Embodiment 2

[0015] Example 2: Using Tango software to optimize the obtained A1 amino acid sequence

[0016] Use Tango software (http: / / tango.crg.es / ) to predict the aggregation-prone region of the amino acid sequence of the A1 candidate clone, and find that there is a strong aggregation-prone region, such as Picture 1-1 , so the region is subjected to point mutations to obtain the A8 antibody, the amino acid sequence of its light chain is shown in SEQ No.1, the amino acid sequence of its heavy chain is shown in SEQ No.3, the corresponding base sequence, and the light chain is shown in SEQ No. 4, the heavy chain is shown in SEQ No.5. The mutated amino acid sequence was re-predicted by Tango software, and it was found that the aggregation-prone region was significantly improved, as shown in Figure 1-2 , 1-3.

Embodiment 3

[0017] Example 3: 293F cells express A8 antibody and detect it by SDS-PAGE

[0018] The mutated base sequence was cloned into the dual promoter vector pVitro2-neo-mcs (InvivoGen), and the 293F cells were transfected with PEI to express the protein. After 5-7 days of expression, the culture supernatant of the 293F cells was treated with ProteinA (GE) was purified, and after the purified protein was concentrated and exchanged, the A8 antibody dissolved in PBS buffer was obtained. After SDS-PAGE electrophoresis detection, it was found that it presented two bands of light chain and heavy chain, such as figure 2 shown.

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Abstract

The invention discloses a human monoclonal antibody against the protein tag Streptactin. After screening and genetic modification, the obtained antibody has good specificity, high affinity, and enhanced anti-aggregation, and is suitable for the purification and detection of the target protein with this tag.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human monoclonal antibody directed against protein tag Streptactin. Background technique [0002] Streptactin (amino acid sequence: WSHPQFEK (GGGS) 2 GGSAWSHPQFEK) is a commonly used tag for protein purification and detection, which can be added to the N-terminal or C-terminal of the target protein for purification and detection of the target protein. The existing antibodies against this sequence are all animal-derived, but there are problems such as poor specificity in detection. Here, we use the human Fab phage display library to screen the human monoclonal antibody against this tag. It can be used for the purification and detection of proteins containing the tag, improving the specificity of detection, etc. Contents of the invention [0003] The purpose of the present invention is to overcome the above defects and provide a human monoclonal antibody against the protein tag S...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13
CPCC07K16/005C07K16/1275C07K2317/92
Inventor 龚睿
Owner 武汉班科生物技术有限公司
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