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Preparation of hepatitis B surface antigen monoclonal antibody

A surface antigen and single-chain antibody technology, applied in the field of bioengineering, can solve the problems of large individual differences, poor detection accuracy, and large batch-to-batch differences of anti-hepatitis B surface antigen monoclonal antibodies

Active Publication Date: 2021-03-19
杭州贤至生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the uncertainty of ascites production in a single mouse and the large individual differences, the obtained anti-HBsAg monoclonal antibody has large differences between batches, which makes the detection accuracy poor.

Method used

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  • Preparation of hepatitis B surface antigen monoclonal antibody
  • Preparation of hepatitis B surface antigen monoclonal antibody
  • Preparation of hepatitis B surface antigen monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0008] Example 1: HBsAg Protein Predominant Epitope Selection

[0009] Taking HBsAg protein as the target antigen, the hydrophilicity and antigenicity of its epitope sequence were analyzed by using the biological software DNAssist2.0, and the A dominant antigenic epitope and the B dominant antigenic epitope were selected. At the same time, the sequence comparison results showed that the selected two dominant antigenic epitopes A and B have high sequence specificity and no obvious homology with other protein sequences.

Embodiment 2

[0010] Example 2: HBsAg protein dominant antigenic epitope tandem

[0011] In order to enhance the stimulation of the selected antigenic epitope to the mouse immune system and facilitate subsequent experiments, the two dominant antigenic epitope sequences of A and B of the HBsAg protein were connected through a flexible fragment (four consecutive glycines) and then repeated four times. , to obtain the amino acid sequence of the recombinant protein.

Embodiment 3

[0012] Embodiment 3: optimize the nucleotide sequence of coding recombinant protein

[0013] In order to increase the expression of recombinant protein in Escherichia coli, under the premise that the amino acid sequence of the recombinant protein remains unchanged, the amino acid sequence encoding the recombinant protein is converted into the corresponding nucleotide sequence according to the preferred codons of Escherichia coli, and the upstream and downstream The nucleotide sequences corresponding to the restriction sites BamHI and EcoRI were added respectively, and were synthesized by General Biosystems Anhui Co., Ltd. The synthesized target gene was cloned in the pMD19-T vector (Bao Bioengineering Dalian Co., Ltd.).

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Abstract

The invention belongs to the technical field of bioengineering. The invention relates to a hepatitis B surface antigen (HBsAg) recombinant protein. An amino acid sequence of the recombinant protein isformed by connecting two dominant antigen epitopes of HBsAg in series, an escherichia coli preferred codon is adopted to convert the amino acid sequence of the recombinant protein into a corresponding nucleotide sequence, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed. Therefore, the expression quantity of the recombinant protein in escherichia coli is increased. The invention also relates to a phage library established by immunizing mice with the recombinant protein, a corresponding recombinant protein single-chain antibody scfv sequenceis obtained through selecting and screening, the obtained scfv sequence is constructed into a complete mouse IgG1 antibody sequence expression vector, a monoclonal antibody is expressed through transient HEK293F cells, the monoclonal antibody is purified, colloidal gold particles are marked respectively, the optimal monoclonal antibody pairing combination is determined through an orthogonal experiment, and therefore, the hepatitis B surface antigen monoclonal antibody has important significance for early diagnosis and prevention and treatment of hepatitis B.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. Specifically, the present invention relates to a new hepatitis B surface antigen recombinant protein, using the recombinant protein to immunize mice to establish a phage library, screening to obtain a specific single-chain antibody scfv sequence, and also involves constructing the obtained scfv sequence into eukaryotic expression The carrier expresses HBsAg monoclonal antibody, and is applied to the early diagnosis of hepatitis B disease. Background technique [0002] Hepatitis B virus (HBV) is a worldwide epidemic infectious disease. About 1 million people die every year from liver failure, liver cirrhosis and hepatocellular carcinoma caused by HBV infection. high incidence of hepatitis. Since the hepatitis B virus was discovered in 1965, hepatitis B surface antigen (HBsAg) has been used as one of the qualitative diagnostic markers of HBV infection. It is not infectious but antigenic. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13C12N15/85
CPCC07K16/082C12N15/85C07K2317/622C07K2317/56C07K2317/64C12N2800/107Y02A50/30
Inventor 吴静李小平洪淑凡朱伟余铭恩
Owner 杭州贤至生物科技有限公司
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