CN109765379B

CN109765379B - Application of mycobacterium tuberculosis protein

Info

Publication number: CN109765379B
Application number: CN201811557374.8A
Authority: CN
Inventors: 曾燚华; 王丽; 罗丹
Original assignee: University of South China
Current assignee: University of South China
Priority date: 2018-12-19
Filing date: 2018-12-19
Publication date: 2021-11-23
Anticipated expiration: 2038-12-19
Also published as: CN109765379A

Abstract

The invention relates to the technical field of biology and discloses application of mycobacterium tuberculosis protein. The invention takes ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in mycobacterium tuberculosis protein as diagnosis antigens, detects corresponding antibodies in serum to be detected through indirect ELISA to judge whether a person to be detected is infected with mycobacterium tuberculosis, and further judges whether the person to be detected suffers from tuberculosis, has high reliability, specificity and sensitivity, and can be used in the field of serological diagnosis of tuberculosis.

Description

Application of mycobacterium tuberculosis protein

Technical Field

The invention relates to the technical field of biology, in particular to application of mycobacterium tuberculosis protein.

Background

Tuberculosis (TB) is a chronic infectious zoonosis caused by infection with the infectious pathogen Mycobacterium Tuberculosis (MTB), one of the most major infectious diseases threatening human health. MTB is mainly classified into human type, bovine type, African type and murine type, and more than 90% of human tuberculosis is caused by infecting human type MTB, and a few are caused by bovine type and African type. MTB can invade all organs of the body, and the affected part is most common in lung, and can also affect other organs such as liver, bone, brain and the like. MTB infection is divided into two states, latent and active. Active tuberculosis refers to a positive sputum smear: namely MTB is discharged from the body, the MTB is propagated actively and has strong toxicity. The origin of TB infection is mainly active tuberculosis patients, which are mainly transmitted through the respiratory tract, but also through the damaged skin, mucous membrane and digestive tract. After an MTB carrier is infected with HIV, the possibility of developing from a latent stage to an active stage is much higher than that of a non-infected HIV carrier, and the disease course is more rapidly developed. There are about 1040 million new TB cases worldwide in 2016 published by the world health organization "2017 global tuberculosis report", of which 167 thousands die from TB. TB is the ninth leading cause of death worldwide, ranked first among infectious diseases, exceeding aids, and has therefore become a significant threat to global public health safety.

At present, the diagnosis methods of tuberculosis mainly comprise the following methods: (1) bacteriological diagnosis: the sputum smear microscopy method is simple, rapid and reliable, but has poor sensitivity; the sputum sample culture is the gold standard for tuberculosis diagnosis, but the traditional solid culture time needs 60-90 days, the liquid culture and drug sensitive test needs about 20 days, the culture time is long, and about 40 percent of sputum smear specimens of adult tuberculosis patients are detected to be negative. (2) Serological diagnosis: the serological diagnosis method for TB mainly utilizes an immunological technique to detect a specific anti-MTB antibody in serum. (3) Tuberculin skin test: the MTB infected organism generates corresponding sensitized lymphocytes, and after PPD is injected again, the organism can generate delayed type hypersensitivity reaction 2-3 days after skin test, and the part of the organism generates red swelling and hard nodules. This test has been used for clinical diagnosis of TB, but it is poorly specific and often cross-reacts with environmental mycobacteria and bacillus calmette-guerin (BCG). (4) MTB nucleic acid amplification detection: the method has good sensitivity, even if bacteria with low copy number in the sample can be detected, but the clinical application has bias, such as improper sample treatment, cross contamination of target sequences or amplification products and the like, which easily causes false negative. (5) IFN- γ release assay: the method is a new method of in vitro immunodetection, and the commercialized diagnostic kit thereof uses EAST6 and CFP10 protein as stimulating antigens and generally adopts an enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay spot method to detect the quantity of T cells generating IFN-gamma and the variation of cytokines in whole blood or peripheral blood mononuclear cells of an organism.

Of all these diagnostic methods, serological detection is an effective method for in vitro diagnosis of TB, and the detection method is simple, rapid, efficient and cheap, and becomes a research hotspot for TB diagnosis in recent years. The method obtains the recombinant antigen through genetic engineering and then uses indirect ELISA to detect the reactivity of the recombinant antigen and the serum of a patient, but the method is difficult to meet the requirements of high sensitivity and specificity. Therefore, the search for MTB antigens with strong specificity and high sensitivity is the key to improve the positive rate of TB serodiagnosis.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel application of ribokinase, fatty acyl-AMP ligase, cystathionine β -lyase and putative protein of mycobacterium tuberculosis, so that the mycobacterium tuberculosis protein can be used as a diagnostic antigen for detecting tuberculosis and has high sensitivity and specificity.

In order to achieve the above purpose, the invention provides the following technical scheme:

the application of one or more than two of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in mycobacterium tuberculosis protein as diagnostic antigen for detecting tuberculosis.

The application of one or more than two of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in mycobacterium tuberculosis protein and its coding gene in preparing diagnostic antigen for detecting tuberculosis.

The application of one or more than two of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in the mycobacterium tuberculosis protein and the coding gene thereof in preparing a kit for detecting tuberculosis.

Wherein the ribokinase is a protein containing a sequence shown in SEQ ID NO. 1, and the protein is 146-196 amino acid sequence protein of a full-length protein (shown in SEQ ID NO. 5) of mycobacterium tuberculosis ribokinase; the fatty acyl-AMP ligase is a protein containing a sequence shown as SEQ ID NO. 2, and the protein is a 1-68 amino acid sequence protein of a mycobacterium tuberculosis fatty acyl-AMP ligase full-length protein (shown as SEQ ID NO. 6); the cystathionine beta-lyase is a protein containing a sequence shown by SEQ ID NO. 3, and the protein is a 43-72 amino acid sequence protein of mycobacterium tuberculosis cystathionine beta-lyase full-length protein (shown by SEQ ID NO. 7); the assumed protein is a protein containing a sequence shown in SEQ ID NO. 4, and the protein is a 1-73 amino acid sequence protein of a total length protein (shown in SEQ ID NO. 8) of the assumed protein of the mycobacterium tuberculosis.

The protein containing the sequence shown in SEQ ID NO:1/2/3/4 of the invention not only comprises the sequence protein shown in SEQ ID NO:1/2/3/4, but also comprises a fusion protein formed by the sequence protein shown in SEQ ID NO:1/2/3/4 and other suitable proteins, such as fusion proteins formed by the sequence proteins shown in SEQ ID NO:1-4 and each other, or a fusion protein formed by a screening label such as histidine; also included are proteins in which one or more amino acids are deleted, added or substituted on the basis of the full-length protein of the putative protein, provided that the deletion, addition or substitution does not involve the sequence region shown in SEQ ID NO. 1-4, e.g., addition of a screening tag such as histidine on the basis of the full-length protein, and ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase, respectively, corresponding to the sequence proteins shown in SEQ ID NO. 1-4.

The invention takes tuberculosis positive serum as target molecule to carry out biological panning on a T7 phage displayed mycobacterium tuberculosis genome DNA library to obtain 19 positive plaques in total, carries out DNA sequencing on PCR amplification products of the plaques, carries out BLAST comparison on the obtained DNA sequences, and shows that the exogenous DNA sequences of the 19 phage comprise 4 different sequences (representative phage P1-P4) which respectively have 100 percent of homology with partial sequences of genes coding ribokinase (corresponding to phage P1), fatty acyl-AMP ligase (corresponding to phage P2), cystathionine beta-lyase (corresponding to phage P3) and hypothetical protein (corresponding to phage P4).

In order to detect whether the representative phages P1-P4 can be specifically combined with TB positive serum, the invention respectively coats the TB positive serum, negative serum and BSA (bovine serum albumin) with an ELISA plate, and adopts an indirect ELISA method to detect the reactivity of the representative phages P1-P4 and the TB positive serum. The results show that the A450 values of the representative phages P1-P4 of the TB-positive serogroup are obviously higher than the A450 values of the TB-negative serogroup and the BSA control group, which indicates that the representative phages P1-P4 can be specifically combined with the TB-positive serum, and also proves that the ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein displayed on the phages can be specifically combined with the TB-positive serum;

in order to confirm that the representative phages P1 to P4 were able to specifically bind to TB-positive serum, the binding of the representative phages P1 to P4 to TB-positive serum was performed by dot immunoblotting. The result shows that obvious spots appear in phage P1-P4 groups and positive control groups, particularly the spot of phage P1 is more obvious, while no spot appears in wild phage control and negative control groups, which shows that thalli P1-P4 can be specifically combined with TB positive serum, wherein phage P1 is more obvious, and shows that ribokinase displayed on the surface of phage P1 is a dominant diagnostic antigen specifically combined with TB positive serum.

In order to verify the potential application value of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and putative protein full-length protein corresponding to the sequence proteins shown in SEQ ID NO. 1-4 in the diagnosis of TB serology, the invention takes tubercle bacillus ribokinase as an object, artificially constructs a prokaryotic expression vector containing a tubercle mycobacterium ribokinase gene (RK gene), and expresses and purifies the recombinant ribokinase. The result shows that the PCR product with the size of 975bp is successfully amplified, the PCR product has 100 percent of homology with the DNA sequence of the RK gene, a prokaryotic expression vector pET-28a (+) -RK is successfully constructed, the recombinant protein containing the histidine tag is expressed and purified, and SDS-PAGE analysis shows that the purified recombinant protein is a single band. Through Western blot identification, the expressed recombinant protein can be specifically combined with an anti-histidine-tag antibody and tuberculosis positive serum;

in order to further verify the potential application value of the full-length protein of the ribokinase in the serological diagnosis of TB, the invention uses indirect ELISA to detect the reactivity of the recombinant ribokinase and different clinical sera, and the result shows that the OD value of the reaction of the recombinant ribokinase and the tuberculosis positive sera is obviously higher than the OD value of the reaction of the recombinant ribokinase and the tuberculosis negative sera. The sensitivity of ELISA reaction is 90%, the specificity is 86%, and the area under ROC curve (AUC) is 0.9741, and the results show that the full-length protein of the ribokinase has better application value in serological diagnosis of TB.

Based on the above test results, the present invention proposes a series of novel applications of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and putative protein of the aforementioned Mycobacterium tuberculosis.

According to the application provided by the invention, the invention also provides a kit for detecting tuberculosis, which comprises a carrier coated with one or more than two of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in mycobacterium tuberculosis protein; or one or more than two diagnostic antigens of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and putatively protein in the mycobacterium tuberculosis protein.

The kit is based on an ELISA method, and can further comprise other corresponding reagents based on the ELISA method, such as enzyme-labeled antigen or antibody, chromogenic substrate, primary antibody, secondary antibody and the like.

According to the technical scheme, ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in the mycobacterium tuberculosis protein are used as diagnosis antigens, and the corresponding antibodies in the serum to be detected are detected through indirect ELISA to judge whether a person to be detected is infected with the mycobacterium tuberculosis or not and further judge whether the person suffers from the tuberculosis, so that the kit has high reliability, specificity and sensitivity, and can be used in the field of serological diagnosis of the tuberculosis.

Drawings

FIG. 1 shows the results of ELISA testing the reactivity of representative phages P1-P4 with TB-positive sera; wherein P1 represents a representative phage P1 whose surface displays a protein sequence encoded by SEQ ID NO. 1, P2 represents a representative phage P2 whose surface displays a protein sequence encoded by SEQ ID NO. 2, P3 represents a representative phage P3 whose surface displays a protein sequence encoded by SEQ ID NO. 3, and P4 represents a representative phage P4 whose surface displays a protein sequence encoded by SEQ ID NO. 4; p < 0.01;

FIG. 2 shows the results of dot immunoblot assays detecting specific binding of representative phages P1-P4 to TB-positive sera; wherein, A1-A4 is the result of representative phages P1-P4; b1 is the result for TB negative sera; b2 is the result of TB positive serum; b3 is the result of control wild-type phage; b4 is blank control result;

FIG. 3 shows the results of the verification of the expression and purification of recombinant ribokinase; wherein a is a PCR identification diagram of a ribokinase recombinant strain, lanes 1-7 sequentially represent randomly picked colony clones; b is SDS-PAGE analysis of the expressed recombinant ribokinase, lanes 1-2 sequentially represent induced and non-induced recombinant bacteria; c SDS-PAGE analysis of the purified recombinant protein, lane 1 purified recombinant protein;

FIG. 4 shows Western blot identification of recombinant ribokinase; wherein, 1 represents that the primary antibody is an antibody against histidine, 2 represents that the primary antibody is tuberculosis positive serum, and 3 represents that the primary antibody is tuberculosis negative serum;

FIG. 5 shows the reactivity of the full-length protein of recombinant ribokinase with clinical serum and ROC curve; wherein a is a reactive result; b is an ROC curve.

Detailed Description

The invention discloses application of mycobacterium tuberculosis protein, and can be realized by appropriately improving process parameters by taking the contents of the mycobacterium tuberculosis protein as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be implemented and applied by modifying or appropriately combining the applications described herein without departing from the spirit, scope and spirit of the invention.

The application of the Mycobacterium tuberculosis protein provided by the present invention is further explained below.

Example 1: ELISA detection test for specific binding of representative phages P1-P4 and TB positive serum

Plates were coated with 100 μ LTB positive serum (100 μ g/mL) (negative serum control and BSA control were established simultaneously) and incubated overnight in a wet box at 4 ℃. Discarding the coating solution, blocking with blocking solution (5% skimmed milk powder) for 2h, washing with PBST for 8 times, and adding 150 μ L of amplified representative phage (3 × 10L) into the wells¹¹pfu, T7 phage displaying coded sequence protein shown in SEQ ID NO 1-4 on the surface), incubating for 2h at 37 ℃, washing 8 times with PBST, adding HRP-labeled anti-T7 phage antibody diluted by 1:1000, incubating for 2h at 37 ℃, washing 8 times with PBST, respectively adding A, B developing solution, keeping away from light at 37 ℃ for 15min, adding stop solution, and measuring the absorbance value at 450nm with an enzyme-labeling instrument (A450).

As shown in fig. 1: the A450 values of the representative phages P1-P4 in the TB positive serogroup are obviously higher than the A450 values of the TB negative serogroup and the BSA control group, which indicates that the sequence proteins shown in SEQ ID NO. 1-4 displayed on the surfaces of the representative phages P1-P4 can be specifically combined with the TB positive serum.

Example 2: dot immunoblot assay for specific binding of representative phages P1-P4 to TB-positive sera

The PVDF membrane, which had been soaked in methanol to be transparent, was washed with TBST (0.5% Tween 20), and 1. mu.L of the amplified representative phage (10) was dropped onto the membrane¹¹pfu, T7 phage displayed on the surface and encoding the sequence protein shown in SEQ ID NO 1-4), drying at room temperature, placing in a refrigerator at 4 ℃ for sealing overnight, washing the membrane for 3 times by TBST, adding preliminarily purified TB positive serum (1:50), incubating for 2h at 37 ℃, washing the membrane for 6 times by TBST, adding goat anti-human IgG antibody (1: 4000) marked by HRP, soaking the membrane for 1h at 37 ℃, washing the membrane for 6 times by TBST, then illuminating by ECL method, developing and fixing. And simultaneously setting a negative control, a blank control, a wild type phage control and a positive control.

To further confirm that the representative phages P1-P4 can specifically bind to TB-positive serum, the binding of the representative phages P1-P4 to TB-positive serum was examined by dot immunoblotting. As shown in fig. 2: the representative phage P1-P4 group and the positive control group have obvious spots, particularly the spot of P1 is more obvious, while the wild phage control group and the negative control group have NO spots, which indicates that the sequence proteins shown in SEQ ID NO. 1-4 displayed on the surfaces of the representative phage P1-P4 can be specifically combined with TB positive serum, wherein P1 is more obvious, and indicates that the ribokinase displayed on the surface of P1 can be specifically combined with TB positive serum, and the ribokinase is a dominant antigen specifically combined with TB positive serum.

Example 3: expression and purification of recombinant RK

Constructing a prokaryotic expression vector containing RK gene, and expressing and purifying the recombinant ribokinase. The results showed that a PCR product of 975bp in size was successfully amplified with 100% homology to the DNA sequence of the RK gene. Successfully constructs a prokaryotic expression vector pET-28a (+) -RK (figure 3a), expresses and purifies a recombinant protein (36kDa) containing a histidine tag (figure 3 b); as shown in FIG. 3c, SDS-PAGE analysis showed that the purified recombinant protein appeared as a single band. These results demonstrate the successful expression and purification of the recombinant ribokinase full-length protein. Through Western blot identification, the recombinant RK can be specifically combined with an anti-histidine-tag antibody and tuberculosis positive serum (figure 4).

Example 4: the recombinant RK has good sensitivity and specificity in serodiagnosis of TB

To further evaluate the potential application value of the full-length protein of ribokinase in serodiagnosis of TB, the reactivity of recombinant RK in example 3 with different clinical sera was tested by indirect ELISA.

The ELISA microplate coated with the recombinant ribokinase full-length protein purified and identified in example 3 was used as an antigen, and clinically collected tuberculosis positive serum and negative serum were used as primary antibodies, respectively, to detect specific IgG in clinical serum by indirect ELISA.

As shown in FIG. 5, the OD value of the recombinant ribokinase reacted with the tuberculosis-positive serum was significantly higher than that of the recombinant ribokinase reacted with the tuberculosis-negative serum. The sensitivity of ELISA reaction is 90%, the specificity is 86%, and the area under ROC curve (AUC) is 0.9741, and the results show that the result of ELISA test is reliable, and the full-length protein of ribokinase has better application value in serological diagnosis of TB.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. The application of one or more than two of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in mycobacterium tuberculosis protein and the coding gene thereof in the preparation of diagnostic antigen for detecting tuberculosis;

the ribokinase is protein with a sequence shown as SEQ ID NO. 1; the fatty acyl-AMP ligase is a protein with a sequence shown as SEQ ID NO. 2; the cystathionine beta-lyase is a protein with a sequence shown in SEQ ID NO. 3; the hypothetical protein is a protein with a sequence shown in SEQ ID NO. 4.

2. The use of claim 1, wherein the ribokinase is a full-length protein of mycobacterium tuberculosis ribokinase; the fatty acyl-AMP ligase is full-length protein of fatty acyl-AMP ligase of mycobacterium tuberculosis; the cystathionine beta-lyase is mycobacterium tuberculosis cystathionine beta-lyase full-length protein; the hypothetical protein is a full-length protein of the hypothetical protein of mycobacterium tuberculosis.

3. The application of one or more than two of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in the mycobacterium tuberculosis protein and the coding gene thereof in preparing a kit for detecting tuberculosis;

4. The use of claim 3, wherein the ribokinase is a full-length protein of Mycobacterium tuberculosis ribokinase; the fatty acyl-AMP ligase is full-length protein of fatty acyl-AMP ligase of mycobacterium tuberculosis; the cystathionine beta-lyase is mycobacterium tuberculosis cystathionine beta-lyase full-length protein; the hypothetical protein is a full-length protein of the hypothetical protein of mycobacterium tuberculosis.

5. A kit for detecting tuberculosis is characterized by comprising a carrier coated with one or more than two proteins of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and hypothetical protein in mycobacterium tuberculosis protein; or one or more than two diagnostic antigens of ribokinase, fatty acyl-AMP ligase, cystathionine beta-lyase and putatively protein in the mycobacterium tuberculosis protein;

6. The kit of claim 5, wherein the ribokinase is a full-length protein of Mycobacterium tuberculosis ribokinase; the fatty acyl-AMP ligase is full-length protein of fatty acyl-AMP ligase of mycobacterium tuberculosis; the cystathionine beta-lyase is mycobacterium tuberculosis cystathionine beta-lyase full-length protein; the hypothetical protein is a full-length protein of the hypothetical protein of mycobacterium tuberculosis.