CN100401066C

CN100401066C - Gold-labeled diagnosis reagent based on combined protein for tubercle bacillus

Info

Publication number: CN100401066C
Application number: CNB2005101032922A
Authority: CN
Inventors: 张灵霞; 吴雪琼
Original assignee: 309th Hospital of PLA
Current assignee: 309th Hospital of PLA
Priority date: 2005-09-23
Filing date: 2005-09-23
Publication date: 2008-07-09
Anticipated expiration: 2025-09-23
Also published as: CN1844929A

Abstract

The present invention relates to a gold-labeled diagnosis reagent of tuberculosis mycobacteria on the basis of combination proteins, which belongs to the technical field of medical immunodiagnosis. B cell antigen determinants of main secretory proteins ESAT6, MPT64, PstS-1 and Ag85B of the tuberculosis mycobacteria are recombined into a group of proteins as antigens and as detection antibodies with specificity for clinical serological diagnosis of tuberculosis. The present invention combines the genes of the B cell antigen determinants of the main secretory proteins of the tuberculosis mycobacteria and expresses and purifies the genes with the method of genetic engineering to obtain a new protein, and the gold-labeled percolation method is used for detecting human serum with tuberculosis. The present invention has the advantages of high sensitivity, simple operation, quick detection, time saving and strong specificity, the sensitization and real infection of the tuberculosis mycobacteria, which are caused by the inoculation of bcg vaccine and the contact with various non tuberculosis mycobacteria in environment, can be differentiated, and the gold-labeled diagnosis reagent can be widely used for clinical serological diagnosis of tuberculosis.

Description

Much's bacillus gold-labeled diagnosis reagent based on combined protein

Technical field

The invention belongs to medical immunology diagnostic techniques field, relate to the new albumen that utilizes the main secretory protein ESAT6 of Much's bacillus, MPT64, PstS-1, Ag85B antigenic determinant to combine and carry out reagent and the application in clinical diagnosis lungy thereof that tubercle mycobaterium detects.

Background of invention

Current, because resistance and anti-multiple medicines is lungy increases, floating population's increase and acquired immune deficiency syndrome (AIDS) and tuberculosis concurrent infection cause tuberculosis to go up in the world.Tuberculosis remains one of the most serious infectious disease in the current whole world, and the whole world has 1/3rd population to have 2,000,000,000 people to infect Much's bacillus approximately, and 1,000 ten thousand new the infecteds are arranged every year approximately, and 3,000,000 people that have an appointment every year die from tuberculosis.The existing tuberculosis patient 5,000,000 of China account for 1/4th of the whole world, and downtrending is slow, and resistant rate is listed in one of " countries and regions that cause caution especially " up to 46%.

Clinical diagnosis lungy is significant for discovery morning lungy and early treatment.The main means of diagnosis of tuberculosis have smear staining microscopy and separation and Culture, tuberculin skin test and the serology detection etc. of pathogen at present.The smear staining microscopy reaches the purpose of detection based on tubercle bacillus acid-fast stain characteristic, and sensitivity is low, the shortcoming of poor specificity (sensitivity is 10-20%) but this method exists.The isolated culture inspection often needs 4-8 week or longer time, often incurs loss through delay clinical diagnosis and treatment.After Much's bacillus is invaded human body, can cause the cellular immunity and the humoral immunity of human body, be the detection method of the easier tubercle bacillus affection of widespread use clinically based on the PPD skin test of cellular immunity, but specificity is relatively poor.Become a kind of important supplementary means of diagnosis of tuberculosis based on the detection of the tuberculosis specific antibody of humoral immunity.In early days, people detect antibody as antigen by the ELISA method with PPD, and this method has certain limitation.One, PPD is a kind of Combination antigen, and a lot of albumen not only Much's bacillus have, and other mycobacteriums also have.Be difficult to differentiate tuberculosis and environment mycobacterial infections person and BCG vaccination sun commentaries on classics person.Two, the ELISA method needs special messenger's operation, the different people operation, and different batch reagent, the result has a great difference, less stable, and also the time is also longer, needs 2-3 hour.The clinical samples difficulty accomplishes to have limited its popularization with to detection.Afterwards, gold mark percolation is applied to the tuberculosis patient detection of antibodies, has shortened the time of detecting (only 10-20 minute) greatly, has reduced artificial factor in the reaction, improved specificity to a certain extent, clinical practice now be mostly kit by this method preparation.But only make antigen with single albumen, just reduced the susceptibility that detects, and the susceptibility itself that golden mark method detects clinical samples just is lower than the ELISA method, and recombinant protein also contains different antigenic determinants, certain cross reaction is arranged, and susceptibility and specificity all need further to improve.

ESAT-6, MPT64, PstS-1, Ag85 are the main secretory proteins of Much's bacillus, can cause the protective immunological reaction of human body.And ESAT6, MPT64 lack in most of environment and Bacille Calmette-Guerin, and the content of PstS-1 in Bacille Calmette-Guerin also is 1/10th of Much's bacillus.1998, Morten etc. reported the B cell antigen determinant of ESAT6, and Thomas etc. have reported the antigenic determinant of MPT64, and the antigenic determinant of PstS-1, Ag85B also is in the news.Contain the PstS-1 intact proteins in the commercially available tuberculosis serum detectable IC-TB card, its susceptibility and specificity all have much room for improvement; Other albumen are not seen the report of making the serology detectable.

Summary of the invention

The objective of the invention is for overcoming the weak point of prior art, a kind of gold-labeled diagnosis reagent of the Much's bacillus based on combined protein is proposed, this reagent sensitivity height, high specificity, and it is easy and simple to handle, quick, do not need staff training and expensive instrument and equipment, can be widely used in clinical clinical serodiagnosis lungy.

Characteristics of the present invention and good result

The present invention makes up the amino acid of the specific B cell antigen determinant of Much's bacillus of the main secretory protein ESAT-6 of Much's bacillus, MPT64, PstS-1, Ag85B.The space structure of the computer software analysis combined protein by routine, select the rational combined protein of space structure, again by conventional genetic engineering means, with combined protein at vivoexpression, purifying and prepare the antibody test gold marked reagent of Much's bacillus.

Utilize combined protein of the present invention can detect at making the tuberculosis specific antibody that antigen produces, increase than the susceptibility and the specificity of single whole Protein Detection by ESAT-6, MPT64, PstS-1, Ag85B albumen as antigen.The main secretory protein B cell antigen of Much's bacillus determinant combined protein and immunogold silver staining use in conjunction can improve susceptibility and the specificity that detects from 2 aspects in theory.Simultaneously, also have quick, the easy-operating characteristics of gold mark percolation, be convenient to clinical application, detect, make a definite diagnosis significant for tuberculosis.Can be widely used in clinical clinical serodiagnosis lungy.

Combined protein among the present invention also can be envelope antigen, preparation mycobacterium tuberculosis antibody ELISA (indirect method) detectable.The antibody of producing among the present invention also can be used as coated antibody, preparation antigen of mycobacterium tuberculosis ELISA (double antibody sandwich method) detectable.

Embodiment

The gold-labeled diagnosis reagent of the Much's bacillus that the present invention proposes prepares embodiment, may further comprise the steps:

1) from document, finds out the amino acid of the special B cell antigen determinant of ESAT-6, MPT64, PstS-1, Ag85B albumen Much's bacillus, make up, space structure by computer software prediction group hop protein, select space structure reasonably to make up, the encoding gene of synthetic combined protein is as follows:

ATG ACA GAG CAG CAG TGG AAT TTC GCG GGT ATC GAG GCC GCG GCA AGC GCA ATCCAG GGA GGC TGT GGC TCG AAA CCA CCG AGC GGT TCG CCT GAA ACG GGC GCC GGTCCA ACG ACC ACG TAC AAG GCC TTC GAT TGG GAC CAG GCC TAT CGC AAG CCA ATC ACCTAT GGT CCC TCG AGT GAC CCG GCATGG GAG CGC AAC GAC CCT ACG CAG CAG AGCTTC CTC GAC CAG GCC AGT CAA CGG GGA CTC GGC GAG GCC CAA GCT CAG CTC AACGCC ATG AAG GGT GAC CTG CAG AGT TCG TTA GGC GCC, these encoding gene two ends contain restriction enzyme site BamH I and HindIII;

2) above-mentioned synthetic genetic fragment is carried out pcr amplification, on extension amplification outcome T carrier, recon is carried out the evaluation of cutting and check order of BamH I and HindIII enzyme, restriction enzyme site with synthetic fragment two ends, downcut the purpose fragment from the T carrier, be cloned on the expression vector PET24b, in e. coli bl21 (DE3), express, be cultured to OD600 and be at 0.6 o'clock and add IPTG to final concentration be the 0.1M abduction delivering, carry out identification and analysis by gel electrophoresis and Western Blotting;

3), adopt gel (the chelating sephrose fast flow of Pharmacia company productions), pass through metal chelate affinity chromatography and come the purifying expressing protein;

4), the human IgG (from through company of HTC of section) that will buy purifying adopts the immune programme for children immunizing rabbit of standard to prepare anti-human IgG antibody (also can comprise IgM, IgA);

5), prepare collaurum by the citric acid reducing process; Promptly 0.01% chlorauride aqueous solution 100ml heated and boiled adds 1% trisodium citrate aqueous solution 4ml under the magnetic agitation, continues heated and boiled solution is till the shiny red; PH to 5.8 is transferred in cooling back, adds 6ug rabbit anti-human igg's ratio in every ml collaurum, with the two mixing, adds an amount of poly-second and alcohol, high speed centrifugation purifying then.

6), through the square formation titration, determine to detect the concentration 2mg/ml of albumen, gets 1ul and drop on the cellulose filter membrane, and the human IgG 1ul that gets 2mg/ml drops on the cellulose filter membrane preparation gold mark diafiltration plate.And outfit sealing damping fluid (the Tris-HCL damping fluid of 0.02mol/LPh7.4 contains 1%BSA, 0.05%Tween-20), lavation buffer solution (the Tris-HCL damping fluid of 0.02mol/L Ph7.4,0.05%Tween-20).Preparation Much's bacillus gold mark diafiltration antibody test reagent.

7), on the basis of Much's bacillus gold mark diafiltration antibody test reagent, be equipped with silver-colored dye liquor reagent such as (silver acetate 0.11%, p-dihydroxy-benzene 0.35%, gum arabics 7.14%); Preparation Much's bacillus gold mark diafiltration (silver is strengthened) antibody test reagent.

Above-mentioned each preparation process is the conventional ripening technology.

Application Example of the present invention:

Embodiment 1: gold mark diafiltration antibody test reagent detects mycobacterium tuberculosis antibody

Get one of gold mark diafiltration plate, add confining liquid 2-4 and drip, treat confining liquid ooze fully do after, add patients serum 2-4 and drip, treat serum ooze fully do after, add lavation buffer solution 2-4 and drip, wait to wash damping fluid ooze fully do after, add gold mark two anti-2-4 and drip, after two impervious the doing, adding lavation buffer solution 2-4 drips, Deng 5-10 minute, treat that left side Quality Control point develops the color fully after, observe the right point and whether develop the color, if take on a red color then positive result, if there is point then negative result does not appear.

Embodiment 2: gold mark diafiltration (silver is strengthened) antibody test reagent detects mycobacterium tuberculosis antibody

Get one of gold mark diafiltration plate, adding confining liquid 2-4 drips, treat confining liquid ooze fully do after, adding patients serum 2-4 drips, treat serum ooze fully do after, add lavation buffer solution 2-4 and drip, wait to wash damping fluid ooze fully do after, adding gold mark two anti-2-4 drips, after two impervious the doing, add lavation buffer solution 2-4 and drip, wait 5-10 minute, after treating that left side Quality Control point develops the color fully, add silver reinforcement reagent 2-4 and drip, after 5-10 minute, observe the right point and whether develop the color, if be the then positive result of black, if there is point then negative result does not appear.

Effect experiment of the present invention is as table 1

The different reagent of table 1 detect the positive rate of different people serum (each 200 parts)

The susceptibility that detects the tuberculosis human blood by the visible reagent of the present invention of last result is 61%, and specificity is 98%, is higher than 48% and 95% of FD mycobacterium tuberculosis antibody diagnosis box.

Claims

1. gold-labeled diagnosis reagent based on the Much's bacillus of combined protein, it is characterized in that, it is that an albumen is made antigen that the B cell antigen determinant of the main secretory protein ESAT6 of employing Much's bacillus, MPT64, PstS-1, Ag85B reconfigures, as the clinical serodiagnostic antibody that detects specifically lungy; The encoding gene of the B cell antigen determinant combined protein of the main secretory protein of described Much's bacillus is:

ATG?ACA?GAG?CAG?CAG?TGG?AAT?TTC?GCG?GGT?ATC?GAG?GCC?GCG?GCA?AGC?GCA?ATCCAG?GGA?GGC?TGT?GGC?TCG?AAA?CCA?CCG?AGC?GGT?TCG?CCT?GAA?ACG?GGC?GCC?GGTCCA?ACG?ACC?ACG?TAC?AAG?GCC?TTC?GAT?TGG?GAC?CAG?GCC?TAT?CGC?AAG?CCA?ATC?ACCTAT?GGT?CCC?TCG?AGT?GAC?CCG?GCA?TGG?GAG?CGC?AAC?GAC?CCT?ACG?CAG?CAG?AGCTTC?CTC?GAC?CAG?GCC?AGT?CAA?CGG?GGA?CTC?GGC?GAG?GCC?CAA?GCT?CAG?CTC?AACGCC?ATG?AAG?GGT?GAC?CTG?CAG?AGT?TCG?TTA?GGC?GCC。