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Preparation method of red fluorescent antibody resisting HBeAg (hepatitis B virus e antigen)

A technology of red fluorescence and antigen antibody, which is applied in the field of preparation of red fluorescent antibody, can solve problems such as low sensitivity, misjudgment of hepatitis B e antigen detection, and non-detection of hepatitis B e antigen

Inactive Publication Date: 2016-06-08
SHENZHEN SHENGBIZHI TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, foreign companies have used chemical chromogenic methods to quantify hepatitis B e antigen. However, chemical chromogenic methods have low sensitivity and cannot detect low-abundance hepatitis B e antigen in blood, which will cause misjudgment in the detection of hepatitis B e antigen.

Method used

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  • Preparation method of red fluorescent antibody resisting HBeAg (hepatitis B virus e antigen)
  • Preparation method of red fluorescent antibody resisting HBeAg (hepatitis B virus e antigen)
  • Preparation method of red fluorescent antibody resisting HBeAg (hepatitis B virus e antigen)

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preparation example Construction

[0047] like figure 1 as shown, figure 1 It is a schematic flowchart of the preparation method of the red fluorescent antibody against hepatitis B e antigen of the present invention. The preparation method of the red fluorescent antibody of anti-hepatitis B e antigen provided by the invention comprises the steps:

[0048]S1: Obtain the mPylRS mutant library lib-mPylRS-pBK of Methanosarcina mazei;

[0049] S2: obtaining NBD-lysine;

[0050] S3: screening to obtain the NBDKRS-pBK plasmid, the NBDKRS-pBK plasmid includes the gene sequence of the pyrrolysine aminoacyl-tRNA synthetase mutant NBDKRS capable of specifically recognizing NBD-lysine;

[0051] S4: Synthesizing the DNA sequence of the anti-hepatitis B e antigen antibody, recombining the DNA sequence of the anti hepatitis B e antigen antibody with the plasmid pBAD to obtain the Escherichia coli expression plasmid anti-HBeAg-4tag-pBAD;

[0052] S5: Mix the NBDKRS-pBK plasmid in step S3 and the anti-HBeAg-4tag-pBAD plasmi...

example 1

[0091] The sensitivity of the fluorescent antibody prepared by the present invention is tested by using low-concentration hepatitis B e antigen as a sample, and the specific steps are as follows.

[0092] S120: Take hepatitis B e antigen, dilute hepatitis B e antigen with phosphate buffered solution (PBS), and prepare the concentration as 0ng / mL, 0.2ng / mL, 0.5ng / mL, 1ng / mL, 1.5ng / mL, 2ng / mL mL, 2.5ng / mL, 10ng / mL, 15ng / mL, 20ng / mL10 gradients of hepatitis B e antigen solution for use.

[0093] S130: Add 10 μL of the anti-hepatitis B e antigen fluorescent antibody and 90 μL of phosphate buffer solution to 11 wells of the microtiter plate, and add 10 μL of non-fluorescence-labeled hepatitis B e antigen fluorescent antibody and phosphate buffer solution to the 11th well. Solution 90μL, respectively named as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and incubated at room temperature for 12h.

[0094] S140: Discard the liquid in the microplate in S130, re-add 100 μL of phosphate buffer to ...

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Abstract

The invention discloses a preparation method of a red fluorescent antibody resisting HBeAg (hepatitis B virus e antigen). Genes of an mPylRS (pyrrolysyl-tRNA synthetase) of a synthesized methanosarcina mazei species and a plasmid pBK are recombined, and mPylRS-pBK is obtained; a mutant library lib-mPyl1RS-pBK is established with mPylRS-pBK as a template; lib-mPylRS-pBK is subjected to electroporation and then screened; NBDKRS-pBK plasmids are screened and recombined; DNA (deoxyribonucleic acid) resisting the HBeAg antibody is recombined with pBAD, and anti-HBeAg-4tag-pBAD is obtained; the plasmidsanti-HBeAg-4tag-pBAD and NBDKRS-pBK are transformed in a competent state, and a biological expression system for preparing the red fluorescent antibody resisting HBeAg is obtained. With the adoption of the preparation method, the red fluorescent antibody resisting HBeAg can be prepared, and the detection sensitivity is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of a red fluorescent antibody against hepatitis B e antigen. Background technique [0002] The abbreviation of hepatitis B is hepatitis B, which is an infectious disease caused by hepatitis B virus infection and is also the main cause of liver cirrhosis and liver cancer. my country is an area with a high incidence of hepatitis B, with more than 93 million infected people. The difficulty of hepatitis B prevention and control is still severe. [0003] The quantification of hepatitis B e antigen is an important basis for doctors to judge the condition and guide treatment. Limited by technology, the detection of hepatitis B e antigen has been in a manual qualitative or semi-quantitative state. Recently, foreign companies have used chemical chromogenic methods to quantify hepatitis B e antigen. However, chemical chromogenic methods have low sensitivity and cannot de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/70C07K16/08
CPCC07K16/082C07K1/14C07K16/08C12N15/65
Inventor 周晓莹胡立夫英格玛·恩伯格埃姆郎·纳瓦什
Owner SHENZHEN SHENGBIZHI TECH DEV CO LTD
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