NP protein for identifying 2019-nCOV antibody and detection tool
阅读说明：本技术 识别2019-nCOV抗体的NP蛋白及检测工具 (NP protein for identifying 2019-nCOV antibody and detection tool ) 是由 徐兴顺 王彬 苗志刚 万波 孙淼 于 2020-04-29 设计创作，主要内容包括：本发明涉及生物检测领域,特别涉及识别2019-nCOV抗体的NP蛋白及检测工具。本发明提供的胶体金检测试剂盒,包括能够识别2019-nCOV抗体的NP蛋白,具有特异性好、灵敏度高、准确度高、方便快捷、易于操作、经济实惠的特点。(The invention relates to the field of biological detection, in particular to NP protein for identifying a 2019-nCOV antibody and a detection tool. The colloidal gold detection kit provided by the invention comprises NP protein capable of identifying the 2019-nCOV antibody, and has the characteristics of good specificity, high sensitivity, high accuracy, convenience, rapidness, easiness in operation, economy and practicability.)
1. An NP protein that recognizes a 2019-nCOV antibody, characterized by having:
(I) an amino acid sequence shown as SEQ ID No. 1; or
(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or
(III) an amino acid sequence which is at least 80% identical to the sequence of (I) or (II).
2. The NP protein of claim 1, wherein the NP protein is encoded by a nucleotide sequence of
(I) a nucleotide sequence shown as SEQ ID No. 2; or
(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID 2; or
(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or
(IV) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence of (I), (II), (III) or (IV).
3. An expression vector comprising the nucleotide of claim 2.
4. A host transformed with the expression vector of claim 3.
5. Use of the NP protein of claim 1, the vector of claim 3 or the host of claim 4 in the preparation of a 2019-nCOV detection reagent or detection tool.
6.2019-nCOV, comprising the NP protein of claim 1, the vector of claim 3, or the host of claim 4.
7.2019-nCOV, wherein the kit is a colloidal gold assay kit comprising the NP protein of claim 1, the vector of claim 3, or the host of claim 4.
8. The assay kit of claim 7, wherein the NP protein of claim 1 has a nominal amount of 18 μ g/ml and a labeling pH of 8.0.
9. The detection tool according to claim 7 or 8, wherein the detection tool comprises a PVC base plate, and the filter paper, the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially fixed on the PVC base plate; the detection tool takes a nitrocellulose membrane as a carrier, takes colloidal gold as a tracer marker, and further comprises confining liquid and resuspension liquid.
10. The method of manufacturing a test kit according to claim 9, comprising the steps of:
step 1: preparing and obtaining a colloidal gold solution;
step 2: adjusting the pH to 8.0, mixing with the NP protein of claim 1, blocking solution, centrifuging, resuspending in resuspension solution, spotting on a conjugate pad, and drying.
Technical Field
The invention relates to the field of biological detection, in particular to NP protein for identifying a 2019-nCOV antibody and a detection tool.
Background
2019A novel coronavirus, also called COVID-19, is a novel coronavirus found in human body in 2019, and has been outbreaked globally at present. The virus has the characteristic of human transmission, and patients infected with the virus are mainly manifested by general aching pain, myasthenia of limbs, fever, dry cough, vomiting and nausea and diarrhea; the symptoms are more serious and are manifested as chest distress, tight breath and dyspnea; few virus carriers did not show any symptoms. Therefore, it is necessary to detect suspected cases with symptoms and people exposed to confirmed cases.
The existing detection method or kit has the problems of long time consumption, high cost, inconvenience, complex operation and the like, so the method for providing the colloidal gold kit for detecting the new coronavirus antibody has important practical significance for further treatment, crowd screening and effectively inhibiting the transmission of the new coronavirus.
Immunochromatography (IC) is a detection method originated in the 80 th decade of the 20 th century. The immune colloidal gold chromatography technology takes a nitrocellulose membrane as a carrier, takes colloidal gold as a tracer marker, the solution to be detected surges (chromatography) on a fiber membrane through capillary action, the marker is continuously (or not) fixed and concentrated on a detection line and shows color (red) reaction through antigen-antibody specific binding, and finally the existence or nonexistence of the substance to be detected is judged through a signal of the detection line. Compared with an ELISA detection method, the method is simple, convenient, rapid and economical, does not need complex instruments, and has the characteristics of direct judgment of results and the like.
Disclosure of Invention
In view of the above, the present invention provides NP proteins and detection tools for recognizing 2019-nCOV antibodies. The detection tool comprises NP protein capable of identifying the 2019-nCOV antibody, and has the characteristics of good specificity, high sensitivity, high accuracy, convenience, rapidness, easiness in operation and economy.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides NP proteins that recognize 2019-nCOV antibodies, having:
(I) an amino acid sequence shown as SEQ ID No. 1; or
(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or
(III) an amino acid sequence which is at least 80% identical to the sequence of (I) or (II).
On the basis of the above-mentioned studies, the present invention provides a nucleotide encoding the NP protein having
(I) a nucleotide sequence shown as SEQ ID No. 2; or
(II) a complementary nucleotide sequence of the nucleotide sequence shown as
(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or
(IV) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III); or
(V) a nucleotide sequence which has at least 80% identity with the nucleotide sequence of (I), (II), (III) or (IV).
The invention also provides an expression vector comprising the nucleotide.
In addition, the present invention also provides a host transformed with the expression vector.
On the basis of the research, the invention also provides application of the NP protein, the vector or the host in preparation of a 2019-nCOV detection reagent or a detection tool.
The invention also provides a 2019-nCOV detection reagent, which comprises the NP protein, the vector or the host.
More importantly, the 2019-nCOV detection tool is a colloidal gold detection kit, and comprises the NP protein, the vector or the host.
In some embodiments of the invention, the NP protein is present in a nominal amount of 18. mu.g/ml and the labeling pH is 8.0.
In some embodiments of the invention, the detection tool comprises a PVC base plate, and the filter paper, the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially fixed on the PVC base plate; the detection tool takes a nitrocellulose membrane as a carrier, takes colloidal gold as a tracer marker, and further comprises confining liquid and resuspension liquid.
In addition, the invention also provides a preparation method of the detection tool, which comprises the following steps:
step 1: preparing and obtaining a colloidal gold solution;
step 2: adjusting pH to 8.0, mixing with the NP protein of
In some embodiments of the invention, the resuspension comprises: from 0.5% (w/w) to 3% (w/w) sucrose and from 0.5% (w/w) to 3% (w/w) BSA.
In some embodiments of the invention, the time of centrifugation is 20 to 60 min.
The colloidal gold detection kit provided by the invention comprises NP protein capable of identifying the 2019-nCOV antibody, and has the characteristics of good specificity, high sensitivity, high accuracy, convenience, rapidness, easiness in operation, economy and practicability.
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.
FIG. 1 shows a plasmid map constructed in example 1 of the present invention; wherein, FIG. 1(A) shows a spectrogram of pET-41a (+) -GST-6His-N plasmid; FIG. 1(B) shows a spectrum of PGEX-4T-2-GST-N-6His plasmid;
figure 2 shows NP proteins that recognize the 2019-nCOV antibody; wherein
FIG. 3 shows a colloidal gold solution;
FIG. 4 shows the results of labeling pH;
FIG. 5 shows the results of the optimal labeling amount of NP protein;
FIG. 6 shows the effect of colloidal gold detection, negative (left); positive (right).
Detailed Description
The invention discloses NP protein for identifying a 2019-nCOV antibody and a detection tool, and a person skilled in the art can use the contents for reference and appropriately improve process parameters to realize the detection. 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 methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The antibody for identifying the 2019-nCOV NP protein and the raw materials and reagents used in the detection tool provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples: