阅读说明：本技术 结核分枝杆菌的蛋白片段、基因片段、检测试剂盒及制备方法与应用 (Protein fragment, gene fragment and detection kit of mycobacterium tuberculosis, and preparation method and application thereof ) 是由 吴利 黄灵 葛章文 陈泽慧 廖萍 李芙蓉 于 2020-07-15 设计创作，主要内容包括：本申请提供了结核分枝杆菌的蛋白片段、基因片段、检测试剂盒及制备方法与应用,属于医学及生物工程领域。本申请提供的结核分枝杆菌的蛋白片段,具有较好的特异性,通过原核表达的方式表达得到的蛋白片段,在保持蛋白片段的抗原特异性的基础上；能快速和大量的获得目的蛋白片段；方便进行检测和分析。结核分枝杆菌的蛋白片段与抗结核分枝杆菌的抗体进行反应,能检测抗体的灵敏度和特异性情况；具备较好的应用价值。(The application provides a protein fragment, a gene fragment, a detection kit and a preparation method and application of mycobacterium tuberculosis, belonging to the field of medical science and bioengineering. The protein fragment of mycobacterium tuberculosis provided by the application has better specificity, and the protein fragment obtained by expression in a prokaryotic expression mode is on the basis of keeping the antigen specificity of the protein fragment; the target protein fragment can be obtained rapidly and massively; convenient detection and analysis. The protein fragment of the mycobacterium tuberculosis reacts with the antibody of the mycobacterium tuberculosis, so that the sensitivity and specificity of the antibody can be detected; has better application value.)

1. Protein fragments of mycobacterium tuberculosis, characterized in that the protein fragments include Rv1419 fragment and Rv2041c fragment; the amino acid sequence of the Rv1419 fragment is shown as SEQ ID NO.1, and the amino acid sequence of the Rv2041c fragment is shown as SEQ ID NO. 2.

2. A gene fragment of mycobacterium tuberculosis, which is characterized by comprising an Rv1419 gene fragment encoding an Rv1419 protein and an Rv2041c gene fragment encoding an Rv2041c protein; the nucleotide sequence of the gene fragment of the Rv1419 is shown as SEQ ID NO.3, and the nucleotide sequence of the gene fragment of the Rv2041c is shown as SEQ ID NO. 4.

3. The kit for detecting the mycobacterium tuberculosis is characterized by further comprising an Rv1419 protein fragment and an Rv2041c protein fragment.

4. The preparation method of the detection kit for the mycobacterium tuberculosis is characterized by comprising the following steps of:

synthesizing an Rv1419 gene fragment and an Rv2041c gene fragment respectively, and connecting the Rv1419 gene fragment and the Rv2041c gene fragment to an expression plasmid respectively through DNA ligase to obtain a recombinant expression plasmid;

and transforming the recombinant expression plasmid into a host cell, and culturing and inducing expression to obtain an Rv1419 protein fragment and an Rv2041c protein fragment.

5. The method for preparing a kit for detecting Mycobacterium tuberculosis as claimed in claim 4, wherein the expression plasmids are pET-21b and pET28 a.

6. The method for preparing a kit for detecting Mycobacterium tuberculosis as claimed in claim 4, wherein the inducer for inducing expression is IPTG, and the final concentration of the inducer is 1 mmol/L.

7. The method for preparing a kit for detecting Mycobacterium tuberculosis as described in claim 6, wherein the time for inducing expression is 450-500 min.

8. The method for preparing a kit for detecting Mycobacterium tuberculosis as claimed in claim 6, wherein the host cell is Escherichia coli, and the host cell is Escherichia coli Arctic Express.

9. The application of the protein fragment of the mycobacterium tuberculosis in detecting the antibody against the mycobacterium tuberculosis is characterized by comprising the steps of detecting whether a sample contains the antibody against the mycobacterium tuberculosis by using the Rv1419 protein fragment and the Rv2041c protein fragment; the targeting sequences of the antibody are an Rv1419 protein fragment and an Rv2041c protein fragment; the amino acid sequence of the Rv1419 protein fragment is shown as SEQ ID NO.1, and the amino acid sequence of the Rv2041c protein fragment is shown as SEQ ID NO. 2.

10. The application of the protein fragment of mycobacterium tuberculosis in detecting the antibody against mycobacterium tuberculosis according to claim 9, wherein the Rv1419 protein fragment and the Rv2041c protein fragment are used for detecting whether the antibody against mycobacterium tuberculosis is contained in the sample or not is detected by an enzyme-linked immunosorbent assay.

Technical Field

The application mainly relates to the field of medical science and bioengineering, in particular to a protein fragment, a gene fragment, a detection kit and a preparation method and application of mycobacterium tuberculosis.

Background

Currently, the main types of mycobacterium tuberculosis antigens for clinically diagnosing tuberculous pleural effusion are: the tuberculosis antibodies 38KD and 16KD, but the sensitivity and specificity of the tuberculosis antibodies can not meet the requirements that the sensitivity of tuberculosis antibody diagnosis proposed by WHO should reach more than 80 percent, and the specificity reaches more than 95 percent.

Content of application

In a first aspect of the application, there is disclosed protein fragments of mycobacterium tuberculosis, including fragments Rv1419 and Rv2041 c; the amino acid sequence of the Rv1419 fragment is shown as SEQ ID NO.1, and the amino acid sequence of the Rv2041c fragment is shown as SEQ ID NO. 2.

The Rv1419 fragment and the Rv2041c fragment are marker antigen fragments of the HRV37 strain of the mycobacterium tuberculosis, so that the Rv1419 fragment and the Rv2041c fragment of the HRV37 strain can be used for better detecting and analyzing the mycobacterium tuberculosis.

In a second aspect of the present application, there is disclosed a gene fragment of mycobacterium tuberculosis, the gene fragment comprising an Rv1419 gene fragment encoding an Rv1419 protein, and an Rv2041c gene fragment encoding an Rv2041c protein; the nucleotide sequence of the gene segment of the Rv1419 is shown as SEQ ID NO.3, and the nucleotide sequence of the gene segment of the Rv2041c is shown as SEQ ID NO. 4.

In a third aspect of the application, a kit for detecting mycobacterium tuberculosis is disclosed, wherein the kit further comprises an Rv1419 protein fragment and an Rv2041c protein fragment.

In the embodiment, by reserving the Rv1419 protein fragment and the Rv2041c protein fragment in the kit, the detection of the sample can be rapidly realized through the Rv1419 protein fragment and the Rv2041c protein fragment.

In a fourth aspect of the present application, a method for preparing a kit for detecting mycobacterium tuberculosis is disclosed, the method comprising the following steps:

synthesizing an Rv1419 gene fragment and an Rv2041c gene fragment respectively, and connecting the Rv1419 gene fragment and the Rv2041c gene fragment to an expression plasmid respectively through DNA ligase to obtain a recombinant expression plasmid;

and (3) transforming the recombinant expression plasmid into a host cell, and culturing and inducing expression to obtain an Rv1419 protein fragment and an Rv2041c protein fragment.

In the embodiment, a large number of Rv1419 protein fragments and Rv2041c protein fragments can be obtained by synthesizing gene fragments encoding the Rv1419 protein fragment and the Rv2041c protein fragment, connecting the gene fragments to an expression plasmid, and inducing expression, so that the method can be applied to detection.

Some embodiments of the foregoing fourth aspect are where the expression plasmids are pET-21b and pET28 a.

By expressing the plasmid, the target protein can be quickly and accurately expressed by induction, and the Rv1419 protein fragment and the Rv2041c protein fragment can be obtained by purification.

In some embodiments of the fourth aspect of the foregoing, the inducer that induces expression is IPTG at a final concentration of 1 mmol/L.

In the examples, the plasmids were induced by IPTG to express the protein of interest in large quantities. The lactose analogue IPTG has the main functions of relieving a repressor on a Lac or Tac promoter and inducing the generation of recombinant protein bFGF, and the influence of the IPTG on the growth of E.coli JM103 is determined by adopting a shake flask experiment. IPTG has no obvious influence on the maximum growth rate and the final cell density of the plasmid-free e.coli JM103, and the results of the maximum growth rate and the final cell density of the plasmid-containing e.coli JM103-pUC18-bFGF are lower than those of plasmid-free JM103 no matter if IPTG is added, which indicates that the cells containing the foreign genes can influence the growth of the host cells no matter whether the foreign genes are expressed or not, but the influence of the foreign genes on the growth of the host cells after expression is more obvious. IPTG is used for inducing and expressing active tryptophanase, the concentration of IPTG has great influence on the activity of the tryptophanase, and low temperature is favorable for activity expression; however, too low a temperature affects the growth rate of the cells, and the fermentation period is prolonged.

In some embodiments of the foregoing fourth aspect, the time for inducing expression is 450-.

In the embodiment, the thalli can be ensured to keep high-efficiency protein expression by the induction of 450-500 min.

In some embodiments of the foregoing fourth aspect, the host cell is escherichia coli, and the host cell is escherichia coli Arctic Express.

In the examples, there are many choices for host cells, and Escherichia coli is selected as the host cell in the present application because it can rapidly obtain a large amount of cells and express a large amount of target proteins during the growth cycle of Escherichia coli.

Coli is also a wide variety of strains, and the use of different strains varies depending on the circumstances, and in this example, it is preferable that E.coli is a strain of Escherichia coli type HB 101.

In a fifth aspect of the present application, the application of the protein fragment of mycobacterium tuberculosis in detecting the antibody against mycobacterium tuberculosis is disclosed, which comprises using Rv1419 protein fragment and Rv2041c protein fragment to detect whether the sample contains the antibody against mycobacterium tuberculosis; the targeting sequences of the antibody are an Rv1419 protein fragment and an Rv2041c protein fragment; the amino acid sequence of the Rv1419 protein fragment is shown as SEQ ID NO.1, and the amino acid sequence of the Rv2041c protein fragment is shown as SEQ ID NO.2

In some embodiments of the foregoing fifth aspect, the detection of whether the sample contains antibodies against mycobacterium tuberculosis using Rv1419 protein fragment and Rv2041c protein fragment is performed by enzyme-linked immunosorbent assay.

Compared with the prior art, the beneficial effect of this application is: the protein fragment of mycobacterium tuberculosis provided by the application has better specificity, and the protein fragment obtained by expression in a prokaryotic expression mode is on the basis of keeping the antigen specificity of the protein fragment; the target protein fragment can be obtained rapidly and massively; convenient detection and analysis. The protein fragment of the mycobacterium tuberculosis reacts with the antibody of the mycobacterium tuberculosis, so that the sensitivity and specificity of the antibody can be detected; has better application value.

Drawings

FIG. 1 is the restriction enzyme electrophoresis of the recombinant plasmid pET-21b-Rv1419 in example 2;

FIG. 2 is the restriction electrophoresis of the recombinant plasmid pET28a-Rv2041c in example 2;

FIG. 3 is an SDS-PAGE experimental picture of the Rv1419 protein expressed by E.coli in Experimental example 3;

FIG. 4 is an SDS-PAGE experimental picture of Rv2041c protein expressed by E.coli in experimental example 3;

FIG. 5 is an SDS-PAGE experimental picture of the Rv1419 protein purified in experimental example 4;

FIG. 6 is an SDS-PAGE experimental picture of the Rv2041c protein purified in experimental example 4;

FIG. 7 is a Western Blot experiment chart of the Rv1419 protein purified in experiment example 4;

FIG. 8 is a Western Blot experiment chart of the Rv2041c protein purified in Experimental example 4.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present application are described in further detail below with reference to examples.