Nanobiosensor and preparation method thereof and mosquito-borne virus antibody detection method using same

Abstract

A nanobiosensor includes a virus-like particle and a luciferase. The virus-like particle is assembled by a structural protein of a mosquito-borne virus. The luciferase forms a fusion protein with the structural protein of the mosquito-borne virus and is displayed on the surface of the virus-like particle. The virus-like particle can bind to specific antibodies against the mosquito-borne virus, and a rapid antibody detection of the mosquito-borne virus can be achieved by immunoassay through the luciferase.

Claims

1. A nano biosensor, comprising: A type of virus particle is composed of a structural protein of a mosquito-borne virus; and a luciferase is formed with the structural protein of the mosquito-borne virus to form a fusion protein and is displayed on the surface of the virus particle. The virus particle does not possess the capsid protein of the mosquito-borne virus. When the mosquito-borne virus is an alphavirus, the luciferase forms a fusion protein with the E2 protein of the structural protein of the alphavirus, and the luciferase is attached to the N-terminus of the E2 protein. When the mosquito-borne virus is a dengue virus, the luciferase forms a fusion protein with the E protein of the structural protein of the dengue virus, and the luciferase is attached to the N-terminus of the E protein. The virus particle can bind to a specific antibody of the mosquito-borne virus, and immunoassay can be performed using the luciferase, thereby achieving rapid detection of the specific antibody of the mosquito-borne virus.

2. The nanobiosensor as claimed in claim 1, wherein the alpha virus is Chikungunya virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Mayaro virus, Ross River virus, Balma Forest virus, Onyonnyon virus, or Sindbis virus.

3. The nano biosensor as claimed in claim 1, wherein the luciferase is a nano luciferase.

4. The nanobiosensor as claimed in claim 1, wherein the nanobiosensor comprises a protein with an amino acid sequence as shown in SEQ ID NO. 3 and SEQ ID NO.

4.

5. The nanobiosensor as claimed in claim 1, wherein the nanobiosensor comprises a protein with an amino acid sequence as shown in SEQ ID NO. 7 and SEQ ID NO.

8.

6. The nanobiosensor as claimed in claim 1, wherein the nanobiosensor comprises a protein with an amino acid sequence as shown in SEQ ID NO. 11 and SEQ ID NO.

12.

7. The nanobiosensor as claimed in claim 1, wherein the nanobiosensor comprises a protein with an amino acid sequence as shown in SEQ ID NO.

15.

8. The nanobiosensor as claimed in claim 1, wherein the nanobiosensor comprises a protein with an amino acid sequence as shown in SEQ ID NO. 18 and SEQ ID NO.

19.

9. The nanobiosensor as claimed in claim 1, wherein the nanobiosensor further includes an additional antigen that forms the fusion protein with the luciferase and is displayed on the surface of the viral particle.

10. A method for fabricating a nano-biosensor, characterized in that a luciferase gene is inserted into a structural protein gene of a mosquito-borne virus, and the capsid protein gene of the mosquito-borne virus is removed, and a baculovirus transduction mosquito system is used to express, assemble, and secrete virus-like particles, wherein the expressed luciferase protein forms a fusion protein with a structural protein of the mosquito-borne virus and is displayed on the surface of the virus-like particles, wherein... These viral particles do not possess the capsid protein of the mosquito-borne virus. When the mosquito-borne virus is an alphavirus, the luciferase system forms a fusion protein with the E2 protein in the structural protein of the alphavirus, and the luciferase is attached to the N-terminus of the E2 protein. When the mosquito-borne virus is a dengue virus, the luciferase system forms a fusion protein with the E protein in the structural protein of the dengue virus, and the luciferase is attached to the N-terminus of the E protein.

11. A method for detecting mosquito-borne virus antibodies, comprising the steps of: (a) providing a nano-biosensor and a magnetic bead capable of binding to human IgG or IgM, wherein the nano-biosensor comprises a virus particle composed of a structural protein of a mosquito-borne virus, and a luciferase forming a fusion protein with the structural protein of the mosquito-borne virus and displayed on the surface of the virus particle, wherein, The virus particles do not have the capsid protein of the mosquito-borne virus. When the mosquito-borne virus is an alpha virus, the luciferase system forms a fusion protein with the E2 protein in the structural protein of the alpha virus, and the luciferase is linked to the N-terminus of the E2 protein. When the mosquito-borne virus is a dengue virus, the luciferase system forms a fusion protein with the E protein in the structural protein of the dengue virus, and the luciferase is linked to the N-terminus of the E protein; (b) the nano-biosensor, the magnetic bead and a sample to be tested are mixed; (c) the magnetic bead is adsorbed and washed with a magnetic rod; and (d) a luciferase action matrix is ​​added and the light signal is detected.

12. The mosquito-borne virus antibody detection method as described in claim 11, wherein the magnetic bead is a protein G-coupled magnetic bead or an anti-human IgM antibody-coupled magnetic bead.

13. The mosquito-borne virus antibody detection method as claimed in claim 11, wherein steps (b) and (c) further include the steps of: (b1) mixing the sample to be tested with the magnetic beads; (c1) adsorbing and washing the magnetic beads with the magnetic rod; (b2) adding the nano biosensor and mixing; and (c2) adsorbing and washing the magnetic beads with the magnetic rod.

14. The mosquito-borne virus antibody detection method as described in claim 11, wherein step (d) involves detecting the light signal using a smartphone.

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