CN1388378A

CN1388378A - Tubercle mycobaterium detecting reagent

Info

Publication number: CN1388378A
Application number: CN 02113890
Authority: CN
Inventors: 鲍朗; 曾献武; 赵明才
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2002-06-17
Filing date: 2002-06-17
Publication date: 2003-01-01

Abstract

The tubercle mycobacterium detecting reagent kit can be used to detect antibody and antigen of tubercle mycobacterium in the clinical serological diagnosis of tuberculosis. The detecting antigens include two specific tubercle mycobacterium antigens ESAT-6 and CFP-10, which are obtained through gene engineering measures and are purified with fusion protein. The detecting antibody is IgG obtained through immunizing animal with the said antigen to produce immunological serum and purifying. The present invention has strong specificity and can distinguish true tubercle mycobacterium infection from sensitization caused by inoculation of bacille calmette guerin and contact with non-tubercle mycobacterium.

Description

Tubercle mycobaterium detecting reagent

1. technical field the invention belongs to medical immunology diagnostics technical field, specifically is detection kit and the application in clinical diagnosis lungy thereof about a kind of Much's bacillus.

2. background of invention tuberculosis is a kind of communicable disease that is caused by Much's bacillus, once occurs being very popular in the whole world.Along with the appearance of effective antituberculotic, taking a turn for the better appears in the tuberculosis popularity.But, surplus in the of nearly ten year over owing to the occur together reasons such as increase of infection and movement of population of appearance, HIV and the tubercle bacillus of tubercle bacillus multi-drug resistant bacterial strain, the tuberculosis epidemic situation is gone up once again, degradating trend has appearred in global tuberculosis situation.WHO announced " the tuberculosis whole world emergency circumstance " in 1993, and WHO in 1998 reaffirms again to contain that action lungy is very urgent.The whole world has 2,000,000,000 people to be subjected to mycobacterium tuberculosis infection approximately at present, existing 2,000 ten thousand tuberculosis patients, and wherein 95% in developing country.Annual neopathy number is 800-1000 ten thousand, and death toll is about 3,000,000, has surpassed the summation of acquired immune deficiency syndrome (AIDS), malaria, diarrhoea, tropical disease death toll.The tuberculosis popularity of China is also very serious always, it is one of the high burden of 22 tuberculosis in whole world country, according to the 4th the national tuberculosis epidemiological random sampling survey resulting estimate that carried out in 2000, China has 4,500,000 active tuberculosis patients approximately, the tuberculosis patient numerical digit occupies the second place of the world, account for 1/4th of global tuberculosis patient, the infectiousness pulmonary tuberculosis patient is about 1,500,000, has every year 12.7 ten thousand people to die from tuberculosis approximately.Tuberculosis has become one of main global disease of current threat human health, also is to cause dead common cause by single infectious agent.The key that reduces tuberculosis mortality rate is early diagnosis, thus formulation that can be accurately and timely rationally, the therapeutic scheme of science.

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 acid-fast stain characteristic that smear staining microscopy method is based on tubercle bacillus reaches the purpose of calibrating, and sensitivity is low, the shortcoming of poor specificity 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.Tulase purified protein derivative (PPD) is the reagent that is widely used in diagnosis of tuberculosis at present, but owing to contain the common antigen molecule of many Mycobacteriums (comprising pathogenic mycobacterium, environment mycobacterium and Bacille Calmette-Guerin) among the PPD, therefore the poor specificity of PPD diagnosis of tuberculosis, actually or can not accurately distinguish the PPD test findings positive because bcg vaccination, with due to the real mycobacterium tuberculosis infection of the sensitization that causes after multiple non tuberculous Mycobacterium in the environment contacts.That the serology detection method has is quick, easy, be convenient to promote, the characteristics that need not special exact instrument are a kind of very potential diagnosis of tuberculosis means, but currently used diagnostic antigen (as PPD, 38KD, 30KD, 14KD, 19KD albumen etc.) exists the shortcoming that specificity is relatively poor, false positive rate is high, is badly in need of a kind of have strong specificity and immunogenic diagnostic antigen.

Mycobacterium tuberculosis ESAT-6-6 is promptly produced by the bacterium secretion in early days at mycobacterium tuberculosis infection, and is discerned by host immune system as a kind of low-molecular-weight secretion property antigen protein.Mycobacterium tuberculosis ESAT-6 antigen protein molecule epi-position is numerous (to have B cell epitope, CD ₄ ⁺, CD ₈ ⁺T cell epitope and DTH epi-position), and only in pathogenic mycobacterium, express, and be not present in Bacille Calmette-Guerin and other non-pathogenic mycobacterium, thereby be a kind of extremely promising tuberculosis specific diagnostic antigen.CFP-10 also is a kind of secretion antigen, and gene and the ESAT-6 of coding CFP-10 are adjacent, are in the same operon, all are positioned at the genomic RD1 section of Much's bacillus, lack equally in Bacille Calmette-Guerin.Coupling ESAT-6 and CFP-10 diagnosis tubercle bacillus affection can improve the susceptibility and the specificity that detect.

3. summary of the invention the present invention is by genetic engineering means, with mycobacterium tuberculosis ESAT-6-6 and CFP-10 albumen at vivoexpression, purifying and prepare the antibody and the antigen detecting agent box of Much's bacillus; And the fusion that ESAT-6 and CFP-10 amalgamation and expression are produced purified after, the antibody and the antigen detecting agent box of preparation Much's bacillus.This kit can detect the antibody and the antigen of Much's bacillus specifically, can distinguish be because bcg vaccination and with sensitization that causes after multiple non tuberculous Mycobacterium in the environment contacts or real m tuberculosis infection, can be used for clinical serodiagnosis lungy.

4. embodiment 1) classify template as with Much's bacillus H37Rv genome sequence, design the primer of ESAT-6 and CFP-10 gene respectively, clone ESAT-6 and CFP-10 gene.2) by genetic engineering means, the ESAT-6 and the CFP-10 gene end of cloning added joining peptide, obtain antigen-4 fusion protein gene.3) change each clone gene over to expression vector, express corresponding protein, carry out identification and analysis by gel electrophoresis and Western Blotting.4) the separation and purification of protein technology of employing standard, each expressing protein of purifying.As: centrifugal, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography etc.5) each expressing protein adopts the immune programme for children immune animal of standard, as: rabbit, cavy, goat etc.Prepare two kinds of corresponding antibody, and its IgG of purifying.6), determine the concentration of envelope antigen (detection albumen), and be equipped with other related reagents that preparation mycobacterium tuberculosis antibody ELISA detects (indirect method) kit through the square formation titration.7), determine the concentration of coated antibody (detection protein antibodies), second antibody (the enzyme mark detects protein antibodies), and be equipped with other related reagents that preparation antigen of mycobacterium tuberculosis ELISA detects (double antibody sandwich method) kit through the square formation titration.

EXAMPLE Example 1:ELISA indirect method detects mycobacterium tuberculosis antibody and gets blood serum sample 0.1ml to be checked, adding has been coated with in the ELISA Plate of fusion antigen, hatches 30 minutes for 37 ℃, and every block of plate is established three hole negative controls, two hole positive controls, a hole blank.Hatch end, discard the liquid in the plate hole, wash plate five times with cleansing solution, button is done.Add anti-human IgG enzyme labelled antibody, fully mixing was hatched 30 minutes for 37 ℃.The same wash plate after, add colour developing liquid 0.1ml, pat mixing, 37 ℃ of lucifuges colour developings 30 minutes.Every hole adds one of stop buffer, pats mixing, surveys with 450nm wavelength microplate reader and reads OD value, result of determination.Embodiment 2:ELISA double antibody sandwich method detects antigen of mycobacterium tuberculosis and gets blood serum sample 0.1ml to be checked, adding has been coated with in the ELISA Plate of fusion antibody (first antibody), hatches 30 minutes for 37 ℃, and every block of plate is established three hole negative controls, two hole positive controls, a hole blank.Hatch end, discard the liquid in the plate hole, wash plate five times with cleansing solution, button is done.Add second antibody (the anti-fusion antibody of rabbit), fully mixing was hatched 30 minutes for 37 ℃.The same washing adds anti-rabbit igg enzyme labelled antibody behind the plate, and fully mixing was hatched 30 minutes for 37 ℃.Wash plate, add colour developing liquid 0.1ml, pat mixing, 37 ℃ of lucifuges developed the color 30 minutes.Every hole adds one of stop buffer, pats mixing, surveys with 450nm wavelength microplate reader and reads OD value, result of determination.

Claims

1. the diagnostic kit of a Much's bacillus is characterized in that adopting specific detection antigen of Much's bacillus and antibody, can be used for clinical serodiagnosis lungy.

2. described specific detection antigen of claim 1 and antibody are two species-specific antigen ESAT-6 of Much's bacillus and the potpourri of CFP-10, and with the immune serum that this hybrid antigen immune animal was produced purified and antibody.

3. described specific detection antigen of claim 2 and antibody also can be a kind of fusions, and this albumen has the antigenic determinant of ESAT-6 and CFP-10 simultaneously, and with the antibody of this protein Preparation.They can combine with antigen with the antibody of mycobacterium respectively specifically.

4. the described antigen protein of claim 2 and claim 3 is to adopt escherichia expression system, expression of recombinant virus system, insect cell expression system, yeast expression system and mammalian cell expression system etc. to express to produce purified obtaining.

5. the described antigen protein of claim 2 and claim 3 also can be used for interior Skin-test of body and the generation of external evoked IFN-r, thereby detects the infection of Much's bacillus.

6. the described Much's bacillus of claim 1 comprises mycobacterium bovis.

7. be envelope antigen with claim 2 and the described albumen of claim 3, the mycobacterium tuberculosis antibody ELISA of preparation detects (indirect method) kit.

8. the described antibody of claim 7 comprises IgG, IgM, IgA.

9. be coated antibody with claim 2 and the described antibody of claim 3, the antigen of mycobacterium tuberculosis ELISA of preparation detects (double antibody sandwich method) kit.