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Recombinant interferon lambda 4 encoded cDNA sequence as well as preparation method and application thereof

A technology of recombinant human interferon and interferon, which is applied in the field of preparation of anti-Hantaan virus (HTNV) drugs, can solve problems such as difficulty in obtaining and hindering the development and application of IFN-λ4

Inactive Publication Date: 2016-12-14
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IFN-λ4 protein is a weakly secreted peptide. IFN-λ4 mainly exists in cells. Individuals carrying the IFNL4-ΔG allele have low expression of IFN-λ4, which is difficult to obtain in large quantities, which seriously hinders the development and application of IFN-λ4.

Method used

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  • Recombinant interferon lambda 4 encoded cDNA sequence as well as preparation method and application thereof
  • Recombinant interferon lambda 4 encoded cDNA sequence as well as preparation method and application thereof
  • Recombinant interferon lambda 4 encoded cDNA sequence as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] [Example 1] Construction of IFN-λ4 expression plasmid pIFNL4

[0031] According to the genome information of the IFN-λ4 (NM_001276254) gene, the codon optimization of the coding region (CDS region, 540 bp in size) was performed, and a 6*His tag and a TEV cleavage site were added to its amino terminal, such as figure 1 The amino acid sequence of the encoded protein is shown in SEQ ID NO:3. Finally, enzyme cutting sites were added at both ends of the EcoR I and xho I. Synthesize the corresponding sequence, and then clone it into the prokaryotic expression vector pET30a to obtain the expression plasmid pIFNL4. The resulting plasmid pIFNL4 was sequenced by Shanghai Sangon Bioengineering Co., Ltd. to prove that its sequence was correct, and the encoded amino acid sequence was consistent with the original sequence.

Embodiment 2

[0032] [Example 2] Transforming plasmid pIFNL4 into competent bacteria BL21 (DE3)

[0033] Adjust the constant temperature water bath to 42°C in advance, and take some crushed ice from the ice maker.

[0034] Take a tube (100 μl) of competent cell suspension from the -80°C refrigerator, thaw it at room temperature, and immediately place it on ice for 5-10 minutes after thawing.

[0035] Add plasmid DNA solution (generally 50~100 ng), the volume does not exceed 10 μl, shake gently, and place on ice for 30 minutes.

[0036] Place in a 42°C water bath for 90 s heat shock (do not shake), then quickly place on ice for 2 min.

[0037] Add 500 μl of LB liquid medium (without kanamycin) preheated at 37°C to the tube, mix gently, and culture with shaking at 37°C for 1 hour to restore the normal growth state of the bacteria and express the plasmid-encoded antibiotic resistance Sex gene (Kana)

[0038] Shake the above-mentioned bacterial liquid, apply 100 μl on a screening p...

Embodiment 3

[0039] [Example 3] SDS-PAGE gel analysis of the expression of IFN-λ4 protein

[0040] Pick a single colony and inoculate it on 5 ml of LB medium (without antibiotics), shake overnight at 200 rpm at 37°C, and then inoculate it with 30 μg / ml Kanamyces at a volume ratio of 1% (V / V). Shake the bacteria at 200 rpm at 37°C until the OD value is between 0.6 and 0.8, add a final concentration of 1 mM inducer IPTG, induce at 37°C for 12 h, then collect the bacteria by centrifugation, and lyse with RIPA Bacteria were resuspended in liquid solution, then lysed by an ultrasonic breaker, centrifuged at 12,000 g for 15 min at a low temperature of 4°C, and the supernatant was collected, and the same amount of supernatant was electrophoresed on two 12% SDS-PAGE, one of which was Stain with Coomassie Brilliant Blue, and the other piece is used to transfer membranes and complete Western blot experiments. Due to the lack of IFN-λ4 antibody, a His tag was specially designed to be added to the 5'...

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Abstract

The invention relates to a recombinant interferon lambda 4 encoded cDNA sequence which can be efficiently expressed in a prokaryotic expression system. The nucleotide sequence of the recombinant interferon lambda 4 encoded cDNA sequence is as shown in SEQ ID NO:1. The invention further provides application of the cDNA sequence in preparing a medicine for treating and / or preventing hemorrhagic fever with renal syndrome caused by hantaan viruses. The invention provides a method and application of recombinant interferon lambda 4. Escherichia coli can be concerted and expression can be induced by using a prokaryotic expression vector containing human interferon lambda 4 encoded cDNA, the optimal induction temperature of a relatively large amount of dissoluble interferon lambda 4 is 18 DEG C, the bacterium shaking speed is 120rpm, the induction time is 4 hours, and the IPTG concentration is 0.1mM. By adopting the product prepared by using the preparation method, basis is made for study on biological activity and action mechanisms of human interferon lambda 4, and the product can be also applied to medicines for treating and / or preventing hemorrhagic fever with renal syndrome caused by hantaan viruses.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant interferon lambda 4 coding cDNA sequence that can be highly expressed in a prokaryotic expression system and its application in preparing anti-Hantaan virus (HTNV) medicines. Background technique [0002] Interferon (Interferon, IFN) exerts antiviral activity by binding to the cognate receptor complex on target cells. The study found that a cytokine is similar to the sequence of type III interferon (IFN-λ), and induces the expression of interferon-stimulated gene (IFN-stimulated gene, ISG) by activating the JAK-STAT pathway, thereby causing antiviral responses, related tissues Name it IFN-λ4 (gene IFN-λ4). [0003] Interferon λ4 (IFN-λ4) is a new member of type III interferon discovered recently. Its nucleotide sequence is similar to IFN-λ3, but only about 30% of the amino acid sequences are identical. At present, interferon λ4 has not been commercialized in the world...

Claims

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Application Information

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IPC IPC(8): C12N15/20C12N15/70C12N1/21A61K38/21A61P31/14A61P13/12
CPCC07K14/555A61K38/21C12N15/70C12N2800/22
Inventor 侯炜李宁陈清宙罗凡熊海蓉冯勇王辉谢林林
Owner WUHAN UNIV
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