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RBD (receptor binding domain) segment in MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and coding gene and application thereof

A protein and residue technology, applied to the RBD fragment in the MERS-CoV virus membrane protein and its encoding gene and application field, can solve the problem of undetermined source and host, and achieve the effect of extensive theoretical guidance value

Active Publication Date: 2015-06-24
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] MERS-CoV belongs to betacoronavirus, but its source and host are still not determined

Method used

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  • RBD (receptor binding domain) segment in MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and coding gene and application thereof
  • RBD (receptor binding domain) segment in MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and coding gene and application thereof
  • RBD (receptor binding domain) segment in MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the preparation of RBD fragment

[0046] 1. Construction of recombinant plasmids

[0047] FastBac TM The dual vector itself does not have a signal peptide secreted extracellularly, and the RBD fragment itself is a secreted protein, so the coding sequence of the signal peptide needs to be connected to the vector in advance, and the signal peptide will be excised during the process of protein expression and secretion in insect cells.

[0048] 1. Synthesize the double-stranded DNA molecule shown in sequence 4 of the sequence listing (the double-stranded DNA molecule shown in sequence 4 of the sequence listing encodes the gp67 signal peptide).

[0049] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0050] F1: 5'-cgg AGATCT ATGCTACTAGTAAATCAG-3';

[0051] R1: 5'-GCG GAATTC GC CGCAAAGGCAGAATGCGC-3'.

[0052] 3. Dige...

Embodiment 2

[0091] Example 2, Preparation of DPP4 Extracellular Region Fragments

[0092] 1. Construction of recombinant plasmids

[0093] 1. Synthesize the double-stranded DNA molecule shown in sequence 7 of the sequence listing.

[0094] 2. Using the double-stranded DNA molecule synthesized in step 7 as a template, perform PCR amplification with a primer pair composed of F3 and R3-1 to obtain a PCR amplification product.

[0095] F3: 5'-cgc ggatcc cAGTCGCAAAACTTACACTCT-3';

[0096] R3-1: 5'-ATACGC GTC GAC CTAatggtggtgatggtggtg AGGTAAAGAGAAACATTGTTTTATG-3'.

[0097] 3. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F3 and R3-2 to obtain a PCR amplification product.

[0098] R3-2: 5'-ATACGC GTC GAC CTA AGGTAAAGAGAAACATTGTTTTATG-3’

[0099]4. Digest the PCR amplified product in step 2 with restriction endonucleases BamHI and SalI, and recover the digested product.

[0100] 5. Digest the recombi...

Embodiment 3

[0119] Example 3, the binding experiment of RBD and DPP4

[0120] 1. Co-expression and pull-down experiments

[0121] 1. The DPP4-WT-P1 generation virus solution prepared in Example 2 and the RBD-His-P1 generation virus solution prepared in Example 1 were respectively infected or co-infected at a density of 2×10 6 cells / mL of Sf9 cell fluid (when infecting separately, the volume ratio of the virus fluid to the cell fluid is 1:100; when co-infecting, the two virus fluids are mixed in equal volumes, and the volume ratio of the mixed virus fluid to the cell fluid is 1:100 ), cultured at 27°C, 110rpm for 72h, then centrifuged at 4000rpm for 5min, and the supernatant was taken.

[0122] 2. Add nickel-coated affinity chromatography column material to the supernatant obtained in step 1, shake at 4°C and 30rpm for 3h, then centrifuge at 4000rpm for 5min, and take the precipitate.

[0123] 3. Add Buffer1 containing 20mM imidazole to the precipitate obtained in step 2 for washing, the...

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Abstract

The invention discloses a RBD (receptor binding domain) segment in a MERS-CoV (Middle East respiratory syndrome coronavirus) membrane protein and a coding gene and application thereof. 1, A protein is as follows: (a) a protein comprising 4th to 243th-site amino acid residues from N terminal of a sequence 1 in a sequence table; or (b) a protein comprising 1th to 243th-site amino acid residues from the N terminal of the sequence 1 in the sequence table; or (c) a protein comprising 4 to 249-site amino acid residues from the N terminal of the sequence 1 in the sequence table; or (d) a protein shown as the sequence 1 of the sequence table. The invention finds that MERS-CoV may have a potential receptor binding domain (Receptor Binding Domain, RBD), and in-depth studies on the RBD help to discover a key target blocking the MERS-CoV invasion, and have important theory reference values to research and development of treatment drugs and preventive vaccines of the MERS-CoV.

Description

technical field [0001] The invention relates to an RBD fragment in the membrane protein of MERS-CoV virus, its encoding gene and application. Background technique [0002] Middle East respiratory syndrome coronavirus (MERS-CoV) was first discovered to infect humans in the Middle East in 2012, and then the diseases caused by this virus infection appeared in several European countries and regions. More than half of infected patients developed severe respiratory disease, and its clinical symptoms were very similar to the disease caused by SARS-CoV that broke out in 2013. Because the disease can be transmitted from person to person, it has attracted great attention around the world. [0003] MERS-CoV belongs to betacoronavirus, but its source and host are still not determined. Similar to other coronaviruses, MERS-CoV uses the membrane protein S glycoprotein on its surface to enter susceptible cells. The S glycoprotein consists of the S1 domain at the N-terminus, the S2 domain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/165C12N15/50C07K16/10A61K39/215A61K39/42A61P31/14C12R1/93
CPCA61K39/215A61K2039/505C07K14/005C07K16/10C12N2770/20022C12N2770/20034
Inventor 王新泉张林琦王年爽史宣玲
Owner TSINGHUA UNIV
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