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Coronavirus

Overall service solution for coronavirus drug screening

[Characteristics of Coronavirus Drug Screening]

High drug screening efficiency (with fluorescence reporter gene), low drug screening cost (1/30 of the screening cost of traditional P3 laboratory), safer drug screening (simulating virus and can be completed by ordinary or P2 laboratory), more complete drug screening scheme (covering all pre clinical evaluation services of drugs) Drug screening is more convenient (animal experiments do not require transgenic mice, and ordinary mice based on modified viruses can meet animal experiments).

[Overall service solution for coronavirus drug screening]

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[Overall Service Solution for Coronavirus Drug Screening] - Pseudovirus

Package of pseudovirus:

Using HIV skeleton plasmid with reporter gene and eukaryotic expression plasmid with Spike gene, the two plasmids were co transfected into 293 cells, and the supernatant (containing HIV Spike chimeric virus and HIV genome with reporter gene) was collected after 2-3 days of transfection.

 

Application scenario:

1. Test whether the spike has the ability to invade the target cell line: use the virus to infect the cell line to be tested, and test the report gene expression in the cell.
2. Test the neutralizing activity of antibody or serum against Spike: use this virus to neutralize and incubate it with the serum or antibody to be tested first. After the incubation, incubate the mixture with VeroE6. By testing the reported gene in VeroE6 cells 48 hours later, and comparing it with cells that have not incubated serum and mock cells, the inhibition efficiency of serum or antibody can be obtained.

 

[Technical Services]:

1. Screening of potential functional mutation sites of coronavirus → sample requirements provided by customers → relevant analysis sequence information provided → delivery content → project report → service cycle 2-3 weeks
2. Mutant construction service → construction of vector pCDNA3.1 → sample requirements provided by customers → provision of mutation site related information → delivery of content sequencing report; Successful construction of recombinant pCDNA3.1 plasmid (concentration>1ug/ul; 50ug) → service cycle 2-4 weeks
3. Pseudovirus packaging service → lentivirus skeleton vector pNL-HIV Luc

 

Packaging scheme

Lentivirus vector is a viral vector system modified on the basis of HIV-1 virus, which can efficiently introduce the target gene (or RNAi) into the primary cells or cell lines of animals and humans. The gene sequence of the S/S mutant of the new coronavirus was cloned and constructed into the eukaryotic expression vector pCDNA3.1, and co transfected with the luciferase reporter plasmid pNL-HIV Luc in HEK-293T cells for the preparation of pseudovirus.

 

Sample requirements provided by customers

The customer provides the sequence of the target gene. If the template is provided, please inform the carrier, stable point of enzyme digestion and other information;

 

Deliverables    1. Test report; 2. Constructed viral recombinant plasmid; 3. Provide at least 1ml virus with titer > 1x108-1x109 TCID50

 

Service cycle 2-4 weeks

 

4. SARS CoV-2 neutralizing antibody detection and screening service

 

Effect cells : HeLa-ACE2 cells/293T-ACE2 cells (stable expression of ACE2)

 

Detection scheme

The pseudovirus SARS CoV-2 nCoV-S infects HeLa-ACE2/293T-ACE2 cells after incubation with antibody or serum to be tested; Chemiluminescence method is used to detect Luciferase luminous value RLU, calculate pseudovirus neutralization inhibition rate of antibody or serum to be tested according to Luciferase luminous value, and evaluate neutralization effect of antibody or serum to be tested.

 

Requirements for samples provided by customers:>100 μ G antibody, concentration>0.2mg/mL, sterile, pH7.1-7.4; Or 100 μ L inactivated serum.
Deliverables: project report
Service cycle: 2-4 weeks

[Overall Service Solution for Coronavirus Drug Screening] - a replication subsystem with exclusive intellectual property rights

For different coronavirus invasions, the replication mechanism should establish a rapid drug screening and evaluation platform with good security and low cost. The new coronavirus replicon stable cell line is a stable cell line containing the new coronavirus replicon based on the replicon system. Compared with the replicator system, the new coronavirus replicon stabilized cell line does not require transfection, which is more convenient and fast. The system contains luciferase reporter gene, which is convenient and quick to test, and the analysis of results is standardized. It can be applied to high-throughput screening of drugs that can inhibit virus replication, so as to reduce the pain point that drug screening must be operated with live virus in P3. At present, Liping is the only commercial company in China that has this resource.

 

Replication daughter cell preparation process:

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Through plasmid transfection, resistance gene screening, cell cloning and reporter gene testing, a cell line that can stably retain and express the replicon plasmid was obtained. The cell line will continue to replicate the new crown genome, simulating the replication steps in the virus life cycle, which can be used to screen drugs that inhibit the virus replication steps.

 

 

Technical service: you only need to provide samples, copy the sub drug screen and give it to us.

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[Overall Service Solution for Coronavirus Drug Screening] - P2 real virus simulates the antiviral effect of drugs most realistically and efficiently.

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As shown in the above figure, three highly pathogenic coronaviruses belong to β At present, the viruses that can be used for new coronation or SARS CoV substitution research are WIV1 and WIV16.

Grade P2: Coronavirus is common to other people, which is used to test the broad-spectrum of drugs; P3, used to test the effectiveness of drugs against new coronavirus and mutant strains; P3: other human coronaviruses, used to test the broad-spectrum of drugs;

 

Technical services: drug screening services under the biosafety platform.

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[Overall service solution for coronavirus drug screening] - P3 animal level

Cell experiment:

After P2 virus screening out candidate drugs, P3 live virus is used to evaluate the drug effect. The following can be tested: Neocrown original strain, South African variant, British variant and Delta strain. The above experimental methods can be used to test the drugs for the whole life cycle of the virus at the cell level. If extensive resistance test is required β Coronavirus activity can also be tested for MERS CoV strain.

Cell experiment:

 

After P2 virus screening out candidate drugs, P3 live virus is used to evaluate the drug effect. The following can be tested: Neocrown original strain, South African variant, British variant and Delta strain. The above experimental methods can be used to test the drugs for the whole life cycle of the virus at the cell level. If extensive resistance test is required β Coronavirus activity can also be tested for MERS CoV strain.

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Animal experiment:

 

Corresponding animal models were established for different coronaviruses in order to more truly evaluate drug efficacy, potential target research and targeted drug design.

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Technical service: drug screening CRO service

 

1. New coronavirus KI-hACE2 mouse model (P3 level): At present, the mouse model (independently developed) that can best simulate the natural infection of new coronavirus can test the effect of drugs on different mutant strains.
2. New coronavirus HFH-HACE2 mouse model (P3 grade): it can evaluate the effect of drugs on different mutants at the early stage of infection.
3. MHV infection model (P2 level): an animal model that can evaluate the effect of drugs on coronavirus in a P2 level environment.
4. MERS virus DPP4 transgenic mouse infection model (P3 level): it can evaluate the effect of drugs on MERS virus.

 

Both parties sign an agreement → the customer provides samples → draws up a plan and implements it → makes an appointment for P3 testing by stages and stages → delivers the technical report

Coronavirus Drug Screening Overall Service Solution Product Catalog

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product

Specifications

Lead time

HeLa-ACE2 stable cell line

1 mL

2-4 weeks

(SARS CoV-2) pseudovirus nCoV-S

1 mL

2-4 weeks

Pseudovirus packaging (service)

/

2-4 weeks

Detection and screening of SARS CoV-2 neutralizing antibody (service)

Qualitative testing

1-2 weeks

 

Quantitative testing

1-2 weeks

Replicon stable cell line

3-5 days

Copy sub drug screen (service)

1 sample

3-5 days

 

2-30 samples

4-7 days

 

More than 30

To be discussed separately

P2 - True virus drug screen (service)

1 sample

5-7 days

 

2-30 samples

7-10 days

 

More than 30

To be discussed separately

P3 - True virus drug screen (service)

1 sample

3-5 days

 

2-30 samples

7-10 days

 

More than 30

To be discussed separately

After-sale service

Online service: telephone service: 24-hour after-sales technical service hotline
Offline service: on site service: provide on-site commissioning reagents and equipment services for customers
Remote support: according to customer needs or technical problems fed back, technical guidance on the use of special conference calls can be provided

Service hotline

Headquarters: building C6, life science and Technology Innovation Park, Qixia District, Nanjing

E-mail:marketing@leading-med. com

Official website:www.leadingmed.cn

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