Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a novel infectious disease that first emerged in Wuhan, China in December 2019 (Wang et al. 2020), especially the total number of cases and deaths has surpassed the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) (Xie et al. 2020). To specify, it has affected at least 190 countries or territories, with 14,647,584 confirmed cases and 608,987 deaths (Worldometer 2020). During the initial outbreak in Wuhan, China, the virus was commonly referred to as the "coronavirus" or "Wuhan coronavirus" or "Wuhan virus". In January 2020, the World Health Organisation recommended "2019 novel coronavirus" (2019-nCov) as the provisional name for the virus, which was in accordance with WHO''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''s 2015 guidance against using geographical locations, animal species, or groups of people in disease and virus names. On 11 February 2020, the name "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2) was accepted. However, SARS-CoV-2 is a descendent of SARS-CoV but was chosen based on the established practice for naming viruses in this species and the relatively distant relationship of SARS-CoV-2 to the SARS-CoV virus in a phylogenetic tree and the distance space. In addition, to avoid confusion with the disease SARS, SARS-CoV-2 sometimes is referred as "the COVID-19 virus" or HCoV-19 (Gorbalenya et al. 2020)
Trang 1UNIVERSITY OF SCIENCE AND TECHNOLOGY OF HANOI
Trang 23.2.1 RNA extraction from samples
3.2.2 cDNA synthesis by reverse transcription enzyme
3.2.3 DNA sequencing and analysis
3.2.4 Phylogenetic tree analysis
3.2.5 Construction of pCR2.1 expression vector carrying E gene and N gene
3.2.6 Transformation of competent E.coli cell with plasmid DNA
3.2.7 Plasmid DNA extraction from E.coli
3.2.8 Restriction enzyme digestion:
3.2.9 Agarose gel electrophoresis
IV/ RESULTS AND DISCUSSIONS
4.1 Results
4.1.1 Insert E gene and N gene into pCR2.1 vector
4.1.2 Select recombinant plasmid carrying E gene and N gene by restriction enzymes 4.1.3 E gene analysis
4.1.4 N gene analysis
Trang 34.2 Discussion
V/ CONCLUSION 17
REFERENCES 18
Trang 4LIST OF ABBREVIATIONS
BLAST Basic Local Alignment Search Tool
DMEM Dulbecco’s modified Eagle’s medium
DPBS Dulbecco's Phosphate Buffered Saline
eNOS Endothelial nitric oxide synthase
iNOS Inducible nitric oxide synthase
MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide
NCBI National Center for Biotechnology Information
NNED N-1-napthylethylenediamine dihydrochloride
NSAIDs Nonsteroidal anti-inflammatory drugs
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
Trang 7ABSTRACT
Coronavirus disease 2019 or COVID-19 is an acute infectious respiratory disease caused by SARS-CoV-2 corona virus It has caused for alarms due to the absence of effective vaccines and antiretroviral therapy, coupled with the ability to spread rapidly since it was first discovered in January 2020 Therefore, the genome sequencing of this virus strain plays a key role in developing a diagnostic kit, assessing the relationship between viral genes and human immune mechanisms as well as developing an effective vaccine treatment
In this study, the E and N gene was cloned from the SARS-CoV-2/human/VIE/NIHE/2020 strain viral RNA by RT-PCR, and inserted into the cloning vector pCR2.1 The recombinant plasmid pCR2.1 vector was confirmed by restriction enzymes and sequencing analysis
The results of sequence analysis of nucleotide (nt) and amino acid (aa) based on the sequencing
of E gene (envelope gene) and N gene (nucleocapsid gene) showed that there were significantly similarity percentage of nt and aa between the SARS-CoV-2 virus strains in this study and other reference SARS-CoV-2 virus strains in the world collected from Genbank The results of phylogenetic tree analysis indicated that the SARS-CoV-2 virus strains is more closely related to SARS 2003 virus strain than to the MERS 2012 virus strain
Key word: SARS-CoV-2, envelope gene, nucleocapsid gene, cloning, sequencing analysis, phylogenetic tree analysis
Trang 8TÓM TẮT
Bệnh virus corona 2019 hay COVID-19 là một bệnh đường hô hấp cấp tính truyền nhiễm gây ra bởi chủng virus corona SARS-CoV-2 Nó đã gây ra sự báo động do không có bất kỳ loại vắc-xin hiệu quả cũng như bất kỳ liệu pháp điều trị bằng thuốc chống virus nào và sự lây lan tương đối nhanh chóng của nó trên toàn cầu, từ lần phát hiện đầu tiên vào đầu tháng 1 năm 2020 Do đó, việc giải mã hệ gen của chủng virus này là yếu tố quan trọng giúp phát triển kit chẩn đoán bệnh, đánh giá sự liên quan giữa gen virus và cơ chế miễn dịch của con người cũng như phát triển vaccine điều trị
Trong nghiên cứu này, gen E và N đã được sao chép từ RNA virus SARS-CoV-2 / human / VIE / NIHE / 2020 bằng RT-PCR và đưa vào vector nhân bản pCR2.1 Plasmid pCR2.1 tái tổ hợp đã được xác nhận bằng enzyme cắt giới hạn và gene sequencing
Kết quả phân tích trình tự nucleotide (nt) và axit amin (aa) dựa trên trình tự gen E (gen envelope)
và gen N (gen nucleocapsid) cho thấy tỷ lệ tương đồng đáng kể giữa nt và aa của chủng vi rút SARS-CoV-2 trong nghiên cứu này và các chủng vi rút SARS-CoV-2 tham chiếu khác trên thế giới được thu thập từ Genbank Kết quả phân tích cây phát sinh gen cho thấy các chủng vi rút SARS-CoV-2 có quan hệ gần gũi hơn với chủng vi rút SARS 2003 so với chủng vi rút MERS
2012
Key word: SARS-CoV-2, envelope gene, nucleocapsid gene, cloning, phân tích trình tự gene, phân tích cây phát sinh
Trang 9During the initial outbreak in Wuhan, China, the virus was commonly referred to as the
"coronavirus" or "Wuhan coronavirus" or "Wuhan virus" In January 2020, the World Health Organisation recommended "2019 novel coronavirus" (2019-nCov) as the provisional name for the virus, which was in accordance with WHO's 2015 guidance against using geographical locations, animal species, or groups of people in disease and virus names On 11 February 2020, the name "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2) was accepted However, SARS-CoV-2 is a descendent of SARS-CoV but was chosen based on the established practice for naming viruses in this species and the relatively distant relationship of SARS-CoV-
2 to the SARS-CoV virus in a phylogenetic tree and the distance space In addition, to avoid confusion with the disease SARS, SARS-CoV-2 sometimes is referred as "the COVID-19 virus"
or HCoV-19 (Gorbalenya et al 2020)
Figure 1: Structure of Coronaviruses
Trang 10Coronaviruses are enveloped, positive-sense, single stranded RNA viruses that can infect a wide range of human and animal species (Chu et al 2020) The corona viral genome encodes four major structural proteins which are the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein All of these components are required to produce a structurally complete viral particle Individually, each protein primarily plays a role in the structure of the virus particle (Schoeman and Fielding 2019a), for instance, the S protein mediates the attachment of the virus to the host cell surface receptors resulting in fusion and subsequent viral entry whereas the M protein is the most abundant protein and defines the shape of the viral envelope The E protein is the smallest of the major structural proteins and participates in viral assembly and budding while the N protein is the only one that binds to the RNA genome and is also involved in viral assembly and budding (Malik 2020)
Table 1: SARS-CoV-2 genome
Trang 11The SARS-CoV-2 genome is similar to typical CoVs, which contains at least ten open reading frames (ORFs) The 5’-terminal two thirds of the genome ORF1a/b encodes two large polyproteins, which form the viral replicase transcriptase complex, coupled with the other ORFs
of SARSCoV-2 on the one- third of the genome encode the same four main structural proteins: spike (S), envelope (E), nucleocapsid (N) and membrane (M) proteins and several accessory proteins with unknown functions which do not participate in viral replication (Malik 2020) In this study, we would like to focus on two structural genes which are E gene encoding for E protein and N gene encoding for N protein of SARS-CoV-2
Envelope protein
Starting with some general information about the E protein, the E protein encoded by the E gene
is the smallest structural protein of SARS-CoV-2 but also the most enigmatic This transmembrane protein has a N-terminal ectodomain and a C-terminal endodomain with ion channel activity (Schoeman and Fielding 2019a) During the replication cycle of SARS-CoV-2,
E gene is abundantly expressed inside the infected cell, but only a small portion is incorporated into the virion envelope (Corse and Machamer 2000) The majority of the protein is localised at the site of intracellular trafficking, where it participates in CoV assembly and budding (Fischer
et al 1998) Based on the importance of E protein in virus production and maturation, we can design the protocol of the real-time RT-PCR assays targeting the E gene to diagnose infectious cases, as well as investigate its capacity in producing effective vaccines for SARS-CoV-2 treatment (Malik 2020)
Nucleocapsid protein
The nucleocapsid (N) protein is encoded by the 9th ORF of SARS-CoV and it is known to be the most abundantly expressed protein of the SARS-CoV-2 The protein is composed of two separate domains, an N-terminal domain (NTD) and a C-terminal domain (CTD) It has been suggested that optimal RNA genome binding requires contribution from both these domains due to a huge amount of positively charged amino acids consisting of N-terminal regions Between these two structural domains, N gene included a highly disordered region, which has been reported to interact with the membrane (M) protein and human cellular hnRNPA1 protein as well as it is also predicted to be a hotspot for phosphorylation Hence, in summary, the N-protein might serve completely different functions during different stages of the viral life cycle Therefore, the
Trang 12investigation of the N gene of SARS-CoV-2 can be used as a component for COVID-19 detection and provide a handsome opportunity to develop an effective vaccine treatment for this disease (Surjit and Lal 2008)
Situation of SARS-CoV-2 research in the world and in Vietnam
Up to 15th July, 2020, there are 163 COVID-19 vaccine candidates being researched and developed: 23 vaccines are tested in humans and 140 candidates are in preclinical phase Besides, the governments have also begun issuing Guidelines for research, development, clinical trials and registration of licenses for the COVID-19 vaccine
Figure 2: Number of circulating COVID-19 therapeutic vaccines Vietnam is currently controlling the COVID-19 epidemic, however, social distancing is just a temporary solution to limit the number of new infectious cases as well as prevent the development and outbreak of COVID-19 There are still many mysteries we need to uncover about the SARS-CoV-2 virus, especially developing an effective vaccine against COVID-19 vaccine Currently, there are 04 domestic manufacturers which are VABIOTECH, POLYVAC, IVAC and NANOGEN are in the process of discovering and developing the COVID-19 vaccine
II/ OBJECTIVES
Trang 13III/ MATERIALS AND METHODS
3.1 Materials
E gene and N gene cloned into pCR2.1 vector was provided by Molecular Microbiology Lab
—Institute of Biotechnology — Vietnam Academy of Science and Technology
E.coli DH5 α strain (InvitrogenTM) was used as a primary host for cloning purpose, which
is engineered to maximize transformation efficiency They are defined by three mutations recA1, endA1 which help plasmid insertion and lacZM15 which enable blue white screening The cells are competent and often used with calcium chloride transformation to insert the desired plasmid
pCR2.1 vector: It has 3'-T overhangs for direct ligation of Taq-amplified PCR products, T7 promoter for in vitro RNA transcription and sequencing, a versatile polylinker with flanking EcoR I sites for easy excision of inserts and a M13 forward and reverse primer sites for sequencing
3.2 Methods:
3.2.1 RNA extraction:
Trizol (Invitrogen) was used to extract RNA of the sample, and extraction protocols followed the manufacturer's standard instructions Add 800uL of Trizol solution into 200uL of virus suspension, vortex and then add 200uL of chloroform and vortex solution for 15 seconds, then incubate at room temperature for 5 minutes The suspension was centrifuged at 12000 rpm for
10 minutes at 40C After centrifugation, transfer the aqueous phase containing RNA into a new eppendorf tube (approximately 500 μL) Precipitating viral RNA by adding 500uL of Isopropanol solution, centrifugation at 12000 rpm for 10 minutes at 40°C Washing the precipitate with 1mL
of Ethanol 70°, centrifuge at 12000 rpm for 10 minutes at 40°C Dissolving the RNA in 30uL of Rnase-free sterile water and confirm the quality of the RNA by Nanodrop measurement The purified RNA would be stored at -20°C
3.2.2 cDNA synthesis by reverse transcription enzyme:
Trang 14The cDNA has been synthesized by reverse transcription enzymes To synthesize cDNA, a SuperScriptTM (Invitrogen) kit was used The components and optimal condition for cDNA synthesis are as follows: 5uL of purified RNA, 4.5uL of RNase-free water, 3uL dNTPs (2.5mM/ type), 2uL of oligo dT primer (200pM / uL), 1uL SuperscriptTM II RNAse H- reverse transcriptase (20U / uL), 0.5uL RNase inhibitor (10U / uL) and 4uL 5x first strand buffer The reaction was performed at 420°C in 60 minutes and then increased to 85°C in 5 minutes
3.2.3 DNA sequencing and analysis:
The nucleotide sequence of studied strain was determined by Sanger's method using ABI prism
3100 Sequencer (Applied Biosystem) with BigDye Terminator v3.1 Cycle Sequencing Kit of Applied cave Biosystems The results of 2-dimensional reading sequences were assembled into complete sequences by ChromasPro, BioEdit (Hall, 2013) coupled with investigating the similarity of gene functions on NCBI data banks by BlastN tool and BlastX E gene and N gene were chosen for further analysis
3.2.4 Phylogenetic tree analysis:
Nucleotide sequences were compared by using the ClustalW software In addition, the analysis
of genetic correlation and evolutionary origin by constructing a phylogenetic tree based on E and
N genes of Sarscov2 strains and reference strains collected from Genbank were performed by MEGA7 tool, which used the Maximum-likelihood similarity algorithm, coupled with a repeat value (bootstrap) 1000 times
3.2.5 Construction of pCR2.1 expression vector carrying E gene and N gene
In order to clone the E gene and N gene into pCR2.1 expression vectors, those two sequences were amplified by PCR, then PCR product and pCR2.1 vector were cut simultaneously with
EcoRI, then purified by agarose gel extraction kit The digested PCR product was then ligated to
the vector by R4-ligase The ligation reaction was performed as below:
Trang 15Table 2: Reaction component for inserting gene into expression vector
Notice: Incubate the reaction at 140C overnight
3.2.6 Transformation of competent E.coli cell with plasmid DNA
Transformation is the process of getting the recombinant vector from a reaction mixture or vector solution into E coli cells To enable the cells to take up circular vector DNA they have to be made competent The method for the preparation of competent cells depends on the transformation method used and transformation efficiency required
E.coli competent cell preparation
1, Inoculate one colony from the LB plate into 2 ml LB liquid medium Shake at 37°C overnight
2 Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask) Shake vigorously at 37°C to OD600 — 0.25-0.3 (usually it takes about 1.5-2 hours)
3 Chill the culture on ice for 15 min Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice
4 Centrifuge the cells for 10 min at 3300g (e.g 4,000 rpm in the Jouan tabletop centrifuge) at 4
°C
5 Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2
6 Keep the cells on ice for 30 min
7 Centrifuge the cells as above