Renaturation method of COVID-19 recombinant protein inclusion bodies

Abstract

The invention discloses a renaturation method of COVID-19 recombinant protein inclusion bodies. According to the renaturation method of the COVID-19 recombinant protein inclusion bodies provided by the invention, after a denatured inclusion body dissolution product is treated by using a nickel chelating column, a renaturation solution containing a denaturant and a reduced concentration gradient ofthe denaturant is used for gradient elution at normal temperature for flow renaturation, so that the method not only reduces the costs, but also significantly shortens the time required for the renaturation of the COVID-19 recombinant protein inclusion bodies, thereby facilitating large-scale renaturation of the COVID-19 recombinant protein inclusion bodies in a short time and at a lower cost; and the protein recovery is high, the purity is high, and the stability is strong, so that a large number of high-quality COVID-19 proteins can be conveniently obtained in a short time and at a lower cost for COVID-19 detection and antibody preparation. The renaturation method of the COVID-19 recombinant protein inclusion bodies provided by the invention is suitable for the renaturation of the COVID-19 RBD recombinant protein inclusion body or the COVID-19 N recombinant protein inclusion body.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a method for renaturation of a novel coronavirus recombinant protein inclusion body. Background technique

[0002] The new type of coronavirus (SARS-CoV-2, or COVID-19) is a new strain of coronavirus first discovered in humans in 2019. It belongs to the same β-coronavirus as SARS coronavirus, and the two have high homology 80%. Different from the common cold or flu, the new coronavirus mainly causes lower respiratory tract infection, causing symptoms such as fever, cough, weakness, and difficulty breathing, posing a serious threat to public health.

[0003] At present, the effective detection methods for the new coronavirus are PCR detection of viral nucleic acid and detection of serum virus antibody IgM / IgG. Existing viral nucleic acid detection kits for PCR detection of viral nucleic acid have high cost and long detection cycle. However, through the detection of serum virus antibo...

Examples

Embodiment 1

[0042] Example 1 Novel coronavirus RBD recombinant protein expression and inclusion body renaturation

[0043] 1. Expression of novel coronavirus RBD recombinant protein

[0044] (1) A BamH I restriction site is introduced into the N-terminus of the novel coronavirus RBD protein, and an Xho I restriction site is introduced into the C-terminus to obtain the novel coronavirus RBD recombinant protein. According to the gene sequence encoding the RBD recombinant protein of the new coronavirus, synthetic primers were designed for PCR amplification. After the PCR amplification product was purified, the PCR amplification product of the gene encoding the RBD recombinant protein of the new coronavirus was obtained. Among them, the full gene sequence of the novel coronavirus RBD protein is shown in SEQ ID NO: 1, the upstream primer for PCR amplification is: acacggatccAGAGTCCAACCAACAGAATC (SEQ ID NO: 3), and the downstream primer is: agagctcgagGAAATTGACACATTTGTTTTTAACC (SEQ ID NO: 4).

...

Embodiment 2

[0059] Example 2 Expression of novel coronavirus N recombinant protein and renaturation of inclusion bodies

[0060] Including the following steps:

[0061] (1) A Sac I restriction site was introduced into the N-terminus of the N protein of the novel coronavirus, and 6*His and Not I restriction sites were introduced into the C-terminus to obtain the recombinant protein of the novel coronavirus N. According to the gene sequence encoding the new coronavirus N recombinant protein, synthetic primers are designed for PCR amplification. After the PCR amplification product is purified, the PCR amplification product of the coding gene of the new coronavirus N recombinant protein is obtained. Among them, the new coronavirus N protein The whole gene sequence is shown in SEQ ID NO:2, the upstream primer for PCR amplification is: tgtcgagctcATGTCTGATAATGGACCC (SEQ ID NO:5), and the downstream primer is: gagtgcggccgcTTAGTGATGGTGGTGATGGTGGGCCTGAGTTGAGTCAGC (SEQ ID NO:6).

[0062] (2) The PC...

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a method for renaturation of a novel coronavirus recombinant protein inclusion body. Background technique

[0002] The new type of coronavirus (SARS-CoV-2, or COVID-19) is a new strain of coronavirus first discovered in humans in 2019. It belongs to the same β-coronavirus as SARS coronavirus, and the two have high homology 80%. Different from the common cold or flu, the new coronavirus mainly causes lower respiratory tract infection, causing symptoms such as fever, cough, weakness, and difficulty breathing, posing a serious threat to public health.

[0003] At present, the effective detection methods for the new coronavirus are PCR detection of viral nucleic acid and detection of serum virus antibody IgM / IgG. Existing viral nucleic acid detection kits for PCR detection of viral nucleic acid have high cost and long detection cycle. However, through the detection of serum virus antibo...

Examples

Embodiment 1

[0042] Example 1 Novel coronavirus RBD recombinant protein expression and inclusion body renaturation

[0043] 1. Expression of novel coronavirus RBD recombinant protein

[0044] (1) A BamH I restriction site is introduced into the N-terminus of the novel coronavirus RBD protein, and an Xho I restriction site is introduced into the C-terminus to obtain the novel coronavirus RBD recombinant protein. According to the gene sequence encoding the RBD recombinant protein of the new coronavirus, synthetic primers were designed for PCR amplification. After the PCR amplification product was purified, the PCR amplification product of the gene encoding the RBD recombinant protein of the new coronavirus was obtained. Among them, the full gene sequence of the novel coronavirus RBD protein is shown in SEQ ID NO: 1, the upstream primer for PCR amplification is: acacggatccAGAGTCCAACCAACAGAATC (SEQ ID NO: 3), and the downstream primer is: agagctcgagGAAATTGACACATTTGTTTTTAACC (SEQ ID NO: 4).

...

Embodiment 2

[0059] Example 2 Expression of novel coronavirus N recombinant protein and renaturation of inclusion bodies

[0060] Including the following steps:

[0061] (1) A Sac I restriction site was introduced into the N-terminus of the N protein of the novel coronavirus, and 6*His and Not I restriction sites were introduced into the C-terminus to obtain the recombinant protein of the novel coronavirus N. According to the gene sequence encoding the new coronavirus N recombinant protein, synthetic primers are designed for PCR amplification. After the PCR amplification product is purified, the PCR amplification product of the coding gene of the new coronavirus N recombinant protein is obtained. Among them, the new coronavirus N protein The whole gene sequence is shown in SEQ ID NO:2, the upstream primer for PCR amplification is: tgtcgagctcATGTCTGATAATGGACCC (SEQ ID NO:5), and the downstream primer is: gagtgcggccgcTTAGTGATGGTGGTGATGGTGGGCCTGAGTTGAGTCAGC (SEQ ID NO:6).

[0062] (2) The PC...

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