阅读说明：本技术 牛细粒棘球蚴病的特异性检测抗原及其应用 (Specific detection antigen of echinococcosis granulosus of cattle and application thereof ) 是由 陈启军 杨娜 姜宁 丁莹莹 桑晓宇 冯颖 陈冉 王新一 于 2020-04-21 设计创作，主要内容包括：本发明公开了牛细粒棘球蚴病的特异性检测抗原及其应用,所述牛细粒棘球蚴病的特异性抗原是如下(a1)或(a2)的蛋白质：(a1)氨基酸序列如SEQ ID.1所示的蛋白质；(a2)将(a1)限定的蛋白质的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加,具有丝氨酸蛋白酶抑制剂作用且可与牛细粒棘球病血清抗体特异性结合的蛋白质。本发明表达了Kazal-type serine protease inhibitor domain-containing protein重组蛋白,并作为试纸条检测线包被抗原；同时,通过时间分辨荧光免疫层析技术结合了免疫标记和免疫层析的特点,以时间分辨荧光微球作为示踪物,制备免疫层析试纸条在临床上操作简单、诊断快速、灵敏度高、特异性强。(The invention discloses a specific detection antigen of echinococcosis granulosus of cattle and application thereof, wherein the specific antigen of echinococcosis granulosus of cattle is a protein (a1) or (a2) as follows: (a1) protein with amino acid sequence shown as SEQ ID.1; (a2) and (b) a protein which has the serine protease inhibitor effect and can be specifically combined with the bovine granulomatosis serum antibody by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a 1). The invention expresses the Kazal-type serine protease inhibitor domain-conjugation protein recombinant protein and is used as a test strip detection line coating antigen; meanwhile, the time-resolved fluorescence immunochromatography technology is combined with the characteristics of immune labeling and immunochromatography, and the time-resolved fluorescence microspheres are used as tracers to prepare the immunochromatography test strip which is simple to operate clinically, rapid in diagnosis, high in sensitivity and strong in specificity.)

1. A protein, such as the protein of (a1) or (a 2):

(a1) protein with amino acid sequence shown as SEQ ID.1;

(a2) and (b) a protein which has the serine protease inhibitor effect and can be specifically combined with the bovine granulomatosis serum antibody by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a 1).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein said nucleic acid molecule is a gene encoding the protein of claim 1, said gene being a DNA molecule of any one of the following:

(b1) DNA molecule with the coding sequence shown in SEQ ID NO. 2;

(b2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) and which encodes the protein of claim 1;

(b3) a DNA molecule having 90% or more homology with the DNA molecule defined in (b1) or (b2) and encoding the protein of claim 1.

4. The use of the protein of claim 1 in the preparation of an immunochromatographic test strip for detecting echinococcosis granulosa in cattle, wherein the test strip comprises a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane having a detection line and a quality control line, and a water absorbent pad fixedly connected to the bottom plate in sequence, the detection line is near one end of the conjugate pad, the quality control line is near one end of the water absorbent pad, and the detection line is coated with the protein of claim 1;

the combination pad is coated with an anti-bovine IgG antibody marked by microspheres;

and a rabbit anti-goat IgG antibody is coated on the quality control line.

5. The use according to claim 4,

the detection antigen is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.1.

6. The use according to claim 4,

the coating concentration of the antigen protein at the detection line is 1 mg/mL.

Technical Field

The invention relates to the technical field of biological detection, in particular to a specific detection antigen of echinococcosis granulosus of cattle and application thereof.

Background

Echinococcosis granulosa is a serious zoonotic disease in humans and animals, and humans or ruminants develop their disease primarily by eating eggs of Echinococcus granulosus by mistake, which is called "worm cancer". At present, research on echinococcosis granulosa diagnosis technology is less, the most common diagnosis method is to examine the polypide by dissection, with the development of molecular technology and immunological technology, there are molecular diagnoses such as LAMP and immunological diagnosis such as ELISA kit method and colloidal gold, but from clinical application, the methods have low detection accuracy and low convenience, and cannot be convenient for basic-level workers to operate and detect. When animals are infected with echinococcus granulosus, the traditional diagnosis method is diagnosis at slaughter, however, during slaughter, the typical saccular tissue may be granuloma caused by inflammation, resulting in wrong diagnosis; serological diagnosis includes colloidal gold immunochromatography and enzyme-linked immunosorbent assay. These methods generally use natural spine balloon fluid, but the use of natural spine balloon fluid has the disadvantages of being susceptible to cross-reactivity with serum from other diseased animals, expensive to prepare, and difficult to commercialize. Although echinococcosis granulosa has been studied in human diagnosis, there are few studies on the diagnosis of animals, and when animals are naturally infected with the disease, the antibody response is not obvious, which is liable to cause diagnosis errors. Therefore, the immune diagnosis of echinococcus granulosus is improved, a reliable antigen is searched, and the establishment of a high-efficiency, accurate, good-specificity and simple diagnosis technology is of great importance.

Disclosure of Invention

Therefore, the invention provides a specific detection antigen for echinococcosis granulosus bovis and application thereof.

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

a protein, such as the protein of (a1) or (a 2):

(a1) protein with amino acid sequence shown as SEQ ID.1;

(a2) and (b) a protein which has the serine protease inhibitor effect and can be specifically combined with the bovine granulomatosis serum antibody by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a 1).

Nucleic acid molecules encoding the proteins described above are also within the scope of the invention.

The nucleic acid molecule is a gene encoding the protein, and the gene is a DNA molecule described in any one of the following:

(b1) DNA molecule with the coding sequence shown in SEQ ID NO. 2;

(b2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) and which encodes the protein of claim 1;

(b3) a DNA molecule having 90% or more homology with the DNA molecule defined in (b1) or (b2) and encoding the protein of claim 1.

The application of the protein in the preparation of an immunochromatographic test strip for detecting echinococcosis granulosa of cattle, wherein the test strip comprises a bottom plate, and a nitrocellulose membrane and a water absorption pad which are sequentially and fixedly connected with a sample pad, a combination pad, a detection line and a quality control line on the bottom plate, the detection line is close to one end of the combination pad, the quality control line is close to one end of the water absorption pad, and the detection line is coated with the protein of claim 1;

the combination pad is coated with an anti-bovine IgG antibody marked by microspheres;

and a rabbit anti-goat IgG antibody is coated on the quality control line.

The detection antigen is a protein with 6 histidines added at the amino terminal of the amino acid sequence shown in SEQ ID.1.

The coating concentration of the antigen protein at the detection line is 1 mg/mL.

The invention has the following advantages:

the invention expresses the Kazal-type serine protease inhibitor domain-conjugation protein recombinant protein and is used as a test strip detection line coating antigen; meanwhile, the time-resolved fluorescence immunochromatography technology combines the characteristics of immune labeling and immunochromatography, and the prepared immunochromatography test strip is simple in clinical operation, rapid in diagnosis, high in sensitivity and strong in specificity by taking the time-resolved fluorescence microspheres as tracers.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a double restriction enzyme-digestion-verified electrophoresis of the recombinant plasmid of Kazal-type serine protein inhibitor domain-ligation protein-pGEX-4T-1 of the present invention;

FIG. 2 is an SDS-PAGE electrophoresis of the Kazal-type serine protein inhibitor domain-conjugation protein-GST recombinant protein of the present invention;

FIG. 3 is a Western-blot identification chart of the Kazal-type serine protein inhibitor domain-conjugation protein-GST recombinant protein of the present invention;

fig. 4 is a schematic structural diagram of the time-resolved fluorescence immunochromatographic test strip of the present invention, in which: 1-PVC base plate; 2-sample pad; 3-a conjugate pad; 4-absorbent paper; 5-nitrocellulose membrane; 6-detection line T; 7-quality control line C;

fig. 5 is a diagram of a specific detection result of the time-resolved fluorescence immunochromatographic test strip of the present invention, in which: 1-echinococcosis granulosa positive serum of cattle, 2-fascioliasis of cattle liver positive serum, and 3-echinococcosis cerebri positive serum;

fig. 6 is a diagram of a specific detection result of the time-resolved fluorescence immunochromatographic test strip of the present invention, in which: 1-4: diluting serum according to the ratio of 1:500, 1:1000, 1:2000 and 1:4000 in sequence; and 5 is a sample diluent.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.