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SARS-CoV-2 full-length cDNA cloning single-copy plasmid and construction method thereof

A sars-cov-2, single-copy technology, applied in the biological field, can solve problems such as unstable proliferation, difficulty in large-genome full-length cDNA, and obstacles to the construction of full-length infectious cDNA of coronaviruses

Pending Publication Date: 2022-01-11
WUHAN INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the coronavirus genome is the largest genome known among RNA viruses, and the replication enzyme gene carried by it is unstable when propagated in bacteria in the form of cDNA clones, and it is difficult to synthesize the full-length cDNA of a large genome in vitro, these make coronavirus Construction of viral full-length infectious cDNA has been greatly hindered [8]

Method used

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  • SARS-CoV-2 full-length cDNA cloning single-copy plasmid and construction method thereof
  • SARS-CoV-2 full-length cDNA cloning single-copy plasmid and construction method thereof
  • SARS-CoV-2 full-length cDNA cloning single-copy plasmid and construction method thereof

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Embodiment 1

[0081] Example 1: Construction of a single-copy plasmid pBacm with a smaller molecular weight and transformation containing a single restriction site

[0082] pBacm plasmid construction strategy such as figure 1 , 2 As shown, the specific steps are as follows:

[0083] (1) Using the pBeloBAC11 plasmid (purchased from the BioVector Plasmid Vector Strain Cell Gene Collection Center) as a template, carry out reverse PCR amplification with primers (SEQ ID NO.4 and NO.5), and the product sequence is shown in SEQ ID NO.3 It shows that both its 5' end and 3' end contain PacI restriction sites.

[0084] (2) Purify the above PCR product and digest it with PacI, then ligate it with T4 DNA ligase to obtain pBacm plasmid (~6.4kb).

[0085] (3) Using the pBacm plasmid as a template, PCR primers (SEQ ID NO.7 and NO.8) back amplify the plasmid macrocircle, the product sequence is shown in SEQ ID NO.6, its 5' end and 3' end Contains NotI and AscI restriction sites, respectively.

[0086]...

Embodiment 2

[0088] Embodiment 2: Construction of screening tool plasmid pBacm-SacB-Kan / Amp

[0089] The construction strategy of plasmid pBacm-SacB-Kan / Amp is as follows image 3 As shown, the specific steps are as follows:

[0090] (1) Using the pK18mob-SacB plasmid (purchased from the BioVector Plasmid Vector Strain Cell Gene Collection Center) as a template, design the 5' end and the 3' end to contain AscI and NotI restriction sites (SEQ ID NO.13 and NO.14 ) to amplify the sucrose lethal gene SacB (SEQ ID NO.12).

[0091] (2) After purifying the PCR product of the above SacB gene, digest it with AscI and NotI.

[0092] (3) Digest the constructed pBacm-AscI-Kan-NotI plasmid with AscI and NotI.

[0093] (4) After purifying the digested products in (2) and (3), ligate them with T4 DNA ligase to obtain the recombinant plasmid pBacm-SacB.

[0094] (5) Using pY44 and pBR322 plasmids as templates, design primers (Kan:SEQ ID NO.16 and NO.17; Amp:SEQ ID NO.19 and NO.20) to amplify Kan (SEQ ...

Embodiment 3

[0104] Embodiment 3: Acquisition of the cDNA of SARS-CoV-2

[0105] The RNA of the stock solution of the SARS-CoV-2 inactivated vaccine developed by Wuhan Institute of Biological Products (see CN202010559132.3) was extracted with QIAamp Viral RNA Mini Kit (purchased from QIAGEN Company), and passed PrimeScript TM DoubleStrand cDNA Synthesis Kit (purchased from Takara Company) was reverse-transcribed to obtain cDNA, which was used as a template for subsequent full-length PCR amplification.

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Abstract

The invention relates to an SARS-CoV-2 full-length cDNA cloning single-copy plasmid and a construction method thereof. Specifically, In-Fusion and Gibson Assembly seamless cloning are combined to perform full-length cDNA synthesis and cloning on an SARS-CoV-2 genome. A single-copy plasmid pBac-mini (named pY1B In the early stage and pBacm In the present stage) which is based on a BAC vector and is stable In replication and smaller In molecular weight is constructed, SARS-CoV-2 full-length cDNA is cloned to the vector, a recombinant single-copy plasmid pBacm-SARS-CoV-2 is constructed, and a Recombineering technology is utilized to perform beneficial base modification, gene knockout and other genetic engineering modification on the full-length cDNA.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a SARS-CoV-2 full-length cDNA clone single-copy plasmid and a construction method thereof. Background technique [0002] Currently, COVID-19 [1] It has been included in the Class B infectious diseases stipulated in the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" and is managed according to Class A infectious diseases. It has the characteristics of strong infectivity, long incubation period, and complicated transmission routes. The clinical symptoms of infected patients are fever, Respiratory symptoms, severe respiratory illness, and pneumonia [2] . [0003] SARS-CoV-2 is a single-stranded positive-strand RNA virus with a genome length of about 29.8 kb, the largest genome among known RNA viruses. The genome structure of SARS-CoV-2 follows the specific genetic features known to coronaviruses (CoVs), with a cap structure at...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/50C12N15/66C12N15/65
CPCC12N15/70C12N15/66C12N15/65C07K14/005C12N2770/20022
Inventor 申硕钱莎莎安欢欢田宇璇周金戈于代冠
Owner WUHAN INST OF BIOLOGICAL PROD CO LTD
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