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Preparation method of anti-HBeAg (hepatitis B virus e antigen) green fluorescent antibody

A green fluorescence, antigen-antibody technology, which is applied in the field of preparation of green fluorescent antibodies, can solve the problems of misjudgment, low sensitivity, and undetectable hepatitis B e-antigen in the detection of hepatitis B e-antigen.

Inactive Publication Date: 2016-04-27
BIO TECH ACADEMY CHINA
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  • Abstract
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  • Claims
  • Application Information

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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 anti-HBeAg (hepatitis B virus e antigen) green fluorescent antibody
  • Preparation method of anti-HBeAg (hepatitis B virus e antigen) green fluorescent antibody
  • Preparation method of anti-HBeAg (hepatitis B virus e antigen) green fluorescent antibody

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preparation example Construction

[0047] The preparation method of the green fluorescent antibody of anti-hepatitis B e antigen provided by the invention comprises the steps:

[0048] S1: Obtain the M.BarkeriPylRS mutation library lib-PylRS-pBK;

[0049] S2: obtaining 7-hydroxycoumarin lysine;

[0050] S3: screening the CouKRS-pBK plasmid, which contains the gene sequence coukrs of the pyrrolysine aminoacyl-tRNA synthetase mutant CouKRS that specifically recognizes 7-hydroxycoumarin;

[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 CouKRS-pBK plasmid in step S3 and the anti-HBeAg-4tag-pBAD plasmid in step S4 according to the first preset ratio, transform by electric shock, introduce into E. coli Top10, and obtain the strain by resistance screening A;

[0053] S6: inoculate and culture the...

example 1

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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 an anti-HBeAg (hepatitis B virus e antigen) green fluorescent antibody. DNA (deoxyribonucleic acid) of (PylRS) for M.Barkeri synthesis is recombined with plasmids pBK through genetic engineering means, and PylRS-pBK is obtained; the PylRS-pBK is taken as a template for establishment of a mutation database lib-PylRS-pBK; the lib-PylRS-pBK is subjected to electroporation and screening; CoukRS-pBK plasmids are screened and recombined; DNA of an anti-HBeAg antibody is recombined with pBAD, and anti-HBeAg-4tag-pBAD is obtained; plasmids anti-HBeAg-4tag-pBAD and CoukRS-pBK are converted into a competent state, and a biological expression system for preparing the anti-HBeAg green fluorescent antibody is obtained. The preparation of the anti-HBeAg green fluorescent antibody is realized, 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 green fluorescent antibody against hepatitis B e antigen. Background technique [0002] Chronic infection of hepatitis B virus (HBV) is the main cause of liver fibrosis, cirrhosis and liver cancer. Nearly 1 million people die every year from diseases such as liver cirrhosis or liver cancer caused by chronic HBV infection. At present, there are nearly 100 million HBV chronically infected people in China, and there are still 100,000 new cases of HBV infection each year. [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 h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/70
CPCC07K16/082C12N15/70C12N2800/101
Inventor 张贯京陈兴明张少鹏高伟明李慧玲潘延超
Owner BIO TECH ACADEMY CHINA
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