Team:PYMS GZ China/Lab notebook


Lab notebook

  • 06/01/2020
    Monday, June 01, 2020 9:00 AM

    Today, we have a talk with Pro. Jie Zhu about SARS-CoV-2 (the severe acute respiratory syndrome coronavirus 2). And then we learned about the biology of the SARS-CoV-2. The disease also is called coronavirus disease 2019 (COVID-19). In March 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic. Coronaviruses is positive sense, single-strand enveloped RNA virus belonging to the family Coronaviridae.

    It can cause illnesses such as the common cold, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). SARS-CoV-2 is dangerous for human beings because it can be passed down from person to person by respiratory droplets predominantly, but aerosolization possible from speaking or singing (especially indoors/prolonged exposure) > fomite. Virus found in respiratory secretions and saliva.

    Zhu says that one of the most urgently needs is how to test the infectivity of the current and future strains of SARS-CoV-2 and immunity against them, as this knowledge is crucial in guiding future research in pandemic control and vaccine development. So we need develop a universal platform for infectivity and immunity evaluation on SARS-CoV-2.

  • 06/02/2020
    Tuesday, June 02, 2020 9:00 AM

    We continue to review article to learn more about the COVID-19.

    Coronavirus is an envelope virus with four structural proteins: spike (S) protein, membrane (M) protein,envelope (E) protein and nucleocapsid (N) protein. S protein is responsible for the virus attachmentand entry to the target cells, which initiate the infection process. S protein plays key roles in induction of protective humoral and cellular immunity during SARS-CoV infection.

    SARS-CoV-2 S promote entry into cells, uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind to human ACE2. Consequently, the S protein was considered as the most attractive target for SARS-CoV vaccine and therapeutic development.

  • 06/03/2020
    Wednesday, June 03, 2020 9:00 AM

    In face of the novel coronavirus pneumonia epidemic, a variety of approaches have been employed to develop prophylactic and therapeutic measures, including whole inactivated vaccine, subunit vaccine, RNA-based vaccine, viral vectored vaccines, monoclonal neutralizing antibodies, fusion inhibitors, most of which was designed to target the S protein.

    However, due to its high infectivity and pathogenicity, SARS-CoV-2 should be handled in bio-safety level 3 (BSL-3) facilities, which has limited the development of anti-viral measures. The accessibility of the live virus stain is another major barrier to develop candidate vaccines and therapeutics.

    Spike from several coronaviruses can be “pseudotyped” onto safer nonreplicative viral particles inplace of their endogenous entry protein, thereby making entry of these particles into cells dependent on Spike. Here, we decided to pseudotype lentiviral particles with Spike.

  • 06/04/2020
    Thursday, June 04, 2020 9:00 AM

    we think pseudotyped particles could be used to conveniently measure Spike-mediated cell entry via fluorescent or luciferase reporters, and to quantify the neutralizing activity of human plasma. So lentiviral construct should contain fluorescent or luciferase or both.

  • 06/05/2020
    Friday, June 05, 2020 9:00 AM

    The major mutation detected to date in the SARS-CoV-2 viral envelope spike protein, which is responsible for virus attachment to the host and is also the main target for host antibodies, is a mutation of an aspartate (D) at position 614 found frequently in Chinese strains to a glycine (G). So we decide to mutant this site.

  • 06/08/2020
    Monday, June 08, 2020 9:00 AM

    By looking for a reagent supplier, we found pLOVE-Luciferase-EGFP is on sale by GenScript. Here’s the map of pLOVE-Luciferase-EGFP, it contains both luciferase and EGFP, which meets the need for our experiment.

  • 06/09/2020
    Tuesday, June 09, 2020 9:00 AM

    we look for the lentiviral packaging helper plasmid psPAX2 on Addgen. Here’s the map of psPAX2, it contains HIV gag, pol, rev and tat. gag, pol and rev is used for package, tat is used for activate transfer plasmid.

  • 06/10/2020
    Wednesday, June 10, 2020 9:00 AM

    We ordered S, S was synthesized, and cloned into pCAGGS vector using seamless cloning by GenScript. here’s the map of pCAGGS-S.

  • 06/11/2020
    Thursday, June 11, 2020 9:00 AM

    The ER retrieval signal (“KxHxx” motif) in the CT domain of SARS-CoV-2 S contributes to its subcellular localization. S-FL mainly localized to the ER, so we decide to remove this part from S.

  • 06/12/2020
    Friday, June 12, 2020 9:00 AM

    We ordered S (1-1254aa), S (1-1254aa) was synthesized, and cloned into pCAGGS vector using seamless cloning by GenScript. Here’s the map of pCAGGS-S (1-1254aa), it contains a shorter S contains C-terminal deletion

  • 06/15/2020
    Monday, June 15, 2020 9:00 AM

    We design D614G mutant primer by Primer 5 and ordered from Qingke (China) here’s the primer sequence.

    S-D614G-F CTGTACCAGGgCGTGAATTGCACCGAGGTGC
    S-D614G-R TGCAATTCACGcCCTGGTACAGCACGGCCACC

  • 06/16/2020
    Tuesday, June 16, 2020 9:00 AM

    We ordered reagents for construct generation, DNA polymerase (Vazyme, P525) of High-fidelity, gel extraction kit (QIAGEN, 28704), DMT enzyme (TransGen, GD111-01), Exnase II (Vazyme, C214)

  • 06/17/2020
    Wednesday, June 17, 2020 9:00 AM

    We ordered cell lines, 293T for packaging and ACE2-293T(cells transfected with human ACE2) for infection.
    Both from ProCell (Wuhan, China).
    Dulbecco’s Modified Eagle Medium (Invitrogen)
    fetal bovine serum (FBS; Gibco, Rockville, MD, USA)
    streptomycin (Invitrogen)

  • 06/18/2020
    Thursday, June 18, 2020 9:00 AM

    We ordered reagents for transfection, lipofectamine 3000 (ThermoFisher Scientific, L3000015),
    OPTI-DMEM (Invitrogen). TransLvTM Lentivirus qPCR Titration Kit (Transgen, FV201).
    ONE-GloTM Luciferase Assay System (Promega, E6120)

  • 06/19/2020
    Friday, June 19, 2020 9:00 AM

    Serum sample (harvested at Day 35 post vaccination by an RBD vaccine was used for infectivity inhibition experiment. Detailed information is provided by Yang et al, Nature 2020 and was a kindly gift from Dr. Jingyun Yang (West China Hospital, Sichuan University).

  • 07/01/2020
    Wednesday, July 01, 2020 9:00 AM

    Nearly all the reagents ordered have been received. We start to do the experiments. Firstly, we need to compare the packaging efficiency of the two forms of S.

    • pCAGGS-S, pCAGGS S (1-1254aa), pLOVE-Luciferase-EGFP: glycerol stock (GenScript) psPAX2: glycerol stock (addgen)
      --> miniprep
      1. some glycerol stock into 6ml LB Broth, 6 ul 100mg/ml Amp.
      2. Agitated at 200rpm, 37℃ for ~12 h
      3. Miniprep: concentration of plamids was measured by Nanodrop, 400 ng/ul for pCAGGS-S ,400 ng/ul for pCAGGS S (1-1254aa) 500 ng/ul for psPAX2 and 800 ng/ul for pLOVE-Luciferase-EGFP

  • 07/03/2020
    Friday, July 03, 2020 9:00 AM

    • Culture Medium preparation (500ml)
      ~445ml DMEM (Invitrogen)+ 50ml FBS+5ml Penicillin&Streptomycin

  • 07/06/2020
    Monday, July 06, 2020 9:00 AM

    • Thaw 293T cells 1. shaking the frozen 293T tube for ~60s
      2. transfer the 293T cell to 15ml tube and then add 3ml Medium, 900 rpm, centrifuge for 5 min
      3. remove supernatant, add 2ml Medium to resuspend 293T cells and count cells by automatic cell counter
      4. 2x106 cells were plated into 100mm culture dish, we plate two, one for S, another for S (1-1254aa)

  • 07/07/2020
    Tuesday, July 07, 2020 9:00 AM

    S and S(1-1254aa) pseudovirus packaging

    • transfection
      we are ready to transfect the 293T cell when it reaches ~80% confluency.
      1. prepare two 15ml tube, add 1ml OPTI-MEM to each tube.
      2. one tube add 40 ul lipofectamine 3000, the other tube add 40 ul P3000 reagent and the following plasmids: 12ug pLOVE-Luciferase-EGFP + 6ug psPAX2 + 2ug S or S (1-1254aa), let stand for 5min
      3. transfer the mix in the first tube to the second tube, gently blow a few times, let stand for 15~20min
      4. remove Medium for 293T and add 3ml fresh Medium, then add the 2ml mixture dropwise to the medium
      5. replace with 10ml fresh Medium about 8h after transfection (about 6:00 PM)

  • 07/09/2020
    Thursday, July 09, 2020 6:00 PM

    Collect the suprenant 48h after transfection, and add 10ml fresh Medium

  • 07/10/2020
    Friday, July 10, 2020 6:00 PM

    • Collect and concentrate viruses
      1. collect the suprenant 72h after transfection
      2. To remove cells in the collected suprenant, suprenant were centriguged by 900rpm, 5min and then filtered through a 0.45 um filter.
      3. Pseudoviruses were concentrated by a centrifugal ultrafiltration device using BD tube ~22000g for 1h
      4. now we see virus clumps gathered at the bottom of the tube, remove the suprenant carefully and add 50ul Medium to dissolve virus.

  • 07/13/2020
    Monday, July 13, 2020 9:00 AM

    • Virus titer determination
      1. virus RNA extraction
      Following the manual of Viral RNA extraction Kit (TAKARA), we extracted the RNA of S and S (1-1254aa) pseudovirus the concentration of S and S (1-1254aa) viral RNA is 80ng/ul and 100ng/ul, respectly.

  • 07/14/2020
    Tuesday, July 14, 2020 9:00 AM

    • Virus titer determination (continued)
      2. cDNA synthesis
      Following the manual of iScript cDNA Synthesis Kit (Bio-rad, 1708890), S and S (1-1254aa) cDNA were synthesis.
      5x iScript Reaction Mix 4ul
      iScript Reverse Transcriptase 1ul
      Nuclease-free water 2.5ul for S, and 5ul for (1-1254aa)
      RNA template 1ug (S or S 1-1254aa)12.5ul for S, and 10 ul for S (1-1254aa)
      total 20ul

      incubate the complete reaction mix in a thermal cycler using the following protocol
      Priming 5 min at 25℃
      Reverse transcription 20 min at 46℃
      RT inactivation 1 min at 95℃
      Optional step Hold at 4℃

      the cDNA was stored at -80℃ when the reation is complete

  • 07/15/2020
    Wednesday, July 15, 2020 9:00 AM

    • Virus titer determination (continued)
      3. qPCR
      Following the manual of TransLvTM lentivirus qPCR Titration Kit (Transgen, FV201), we measured the titer of S and S (1-1254aa).

      • Here’s the S-FL and S (1-1254aa) amino acid sequence

      S-FL (S-full length, 1-1273)
      MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
      1255-1273 were labeled green

      S (1-1254aa)
      MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKG

      here’s the titer of S-FL and S (1-1254aa)

      S-FLS (1-1254aa)
      titer33325.4205270384.017

  • 07/20/2020
    Monday, July 20, 2020 9:00 AM

    Due to the higher package efficiency of S (1-1254aa), we aim to use the S (1-1254aa) in subsequent experiments.

    • S mutant—S D614G
      As many paper described, D614G has much higher infection efficiency, so we choose to engineer this site.
      1. PCR
      1ul primer F (10uM) + 1ul primer R (10uM) + 25ul 2X PCR reaction mix (Vazyme) + 0.5ul pCAGGS-S plasmid (1ug/ul) + 22.5uL nuclease free water

      step 1 95℃ 5min
      step 2 95℃ 30sec
      step 3 56℃ 30sec
      step 4 72℃ 4min
      repeat step 2 to step 4 for 28 cycles
      72℃ 10min
      4℃ hold


      2. Gel electrophoresis
      1% agarose gel 120V 30min

      the right size for S and S-D614G should be ~8500bp
      However, there’s no band is at or around ~8500bp, maybe the template DNA is too much and anneal temperature is low, we’ll try another reaction condition tomorrow.

  • 07/21/2020
    Tuesday, July 21, 2020 9:00 AM

    Due to the failure of PCR, we improve the reation condition—less template DNA and higher anneal temperature.

    • S mutant—S D614G
      1. PCR
      1ul primer F (10uM) + 1ul primer R (10uM) + 25ul 2X PCR reaction mix (Vazyme) + 0.2ul pCAGGS-S plasmid (1ug/ul) + 22.8uL nuclease free water

      step 1 95℃ 5min
      step 2 95℃ 30sec
      step 3 58℃ 30sec
      step 4 72℃ 4min
      repeat step 2 to step 4 for 28 cycles
      72℃ 10min
      4℃ hold

      2. Gel electrophoresis
      1% agarose gel 120V 30min

      We finally got the right size for PCR products. so we then need to purify the DNA from gel.

      2. Gel extraction
      Following the manual of QIAquick Gel Extraction Kit (Qiagen, 28704), we purified the S and S-D614G DNA, the product were stored at -20℃

  • 07/22/2020
    Wednesday, July 22, 2020 9:00 AM

    • S mutant—S D614G (continued)
      3. DMT
      we need to remove the remaining template DNA, so we used DMT enzyme which can digest methylated DNA, the template DNA from Ecoli. is methylated, but the DNA amplified by PCR is not methylated.

      15ul gel extraction product + 1ul DMT enzyme + 2ul 5Xbuffer + 2 ul ddH2O = total 20 ul
      37℃ 1h
      80℃ 20min
      4℃ hold
      The purified and digested S and S-D614G are stored at -20℃

  • 07/23/2020
    Thursday, July 23, 2020 9:00 AM

    As the S and S-D614G are still linearized now, we need to make them be circled. So we need to use Exnase II (C214, Vazyme) to cut the overlapped DNA segment and then recombined to circled DNA

    • S and S-D614G construct
      1. Exanse II
      0.5ul recombination Exanse II enzyme + 1ul 5X buffer + 3.5ul DMT-digested product=total 5 ul

      37℃ 30min
      4℃ hold

      2. Transform
      we then transform the 5ul recombined product into Stbl3 (TransGen)
      a. thaw stbl3 component cell (in the tube) on ice for 2-5min carefully remove the caps from the tube and set aside for later use
      b. add 5ul construct into each tube mix the cells by gently stirring the mixture with a pipette tip three times. DO NOT mix by pipetting up and down
      c. cap the tubes and incubate the cells on ice for 30mins
      d. incubate the tube of cells at 42℃ in a water bath. DO NOT mix or shake incubate the tube on ice for 2min
      e. Place the tube on the benchtop, then add 250ul of LB medium
      f. cap the tube and secure at 45 angles in the incubator and shake at 37℃, 225 rpm for 1h
      g. plate 50-100ul step 9 product on pre-warmed LB-ampicillin plate by using a sterile wiper. incubate at 37℃ O/N

  • 07/24/2020
    Friday, July 24, 2020 9:00 AM

    There are many clones grow on the plates (see below)

    so, we need to expand the single clones by clone selection

    • clone selection
      1. we select two from each plate using sterile tips to 10ml tube each tube contains 6ml LB medium and 6ul 100mg/ml Amp
      2. cap the tube and secure at 45 angles in the incubator and shake at 37C, 225 rpm for 1h g. plate 50-100uL step 9 product on pre-warmed LB-ampicillin plate by using a sterile wiper, incubate at 37℃ O/N

  • 07/27/2020
    Monday, July 27, 2020 9:00 AM

    Extract the plamids DNA of single clones according to Mini Prep protocol then we send the plamids to company for sequencing.

  • 07/31/2020
    Friday, July 31, 2020 9:00 AM

    Analysis the sequencing result, we found that we luckily got one D614G mutant clone (below). the site 614 of WT S is GAC(coding D, short for Asp amino acid), however, this site of mutant S-D614G is GGC (coding G, short for Gly amino acid).

  • 08/13/2020
    Thursday, August 13, 2020 9:00 AM

    we prepare package S and S-D614G pseudovirus

    • Culture Medium preparation
      ~445ml DMEM (Invitrogen)+ 50ml FBS+5ml Penicillin&Streptomycin
    • Thaw 293T cells
      1. shaking the frozen 293T tube for ~60s
      2. transfer the 293T cell to 15ml tube and then add 3ml Medium, 900 rpm, centrifuge for 5 min
      3. remove supernatant, add 2ml Medium to resuspend 293T cells and count cells by Automatic cell counter
      4. 2x106 cells were plated into 100mm culture dish, we plate two, one for S, another for S-D614G

  • 08/15/2020
    Saturday, August 15, 2020 9:00 AM

    S(1-1254aa) D614G pseudovirus packaging, this schematic below show how we get virus.

    • transfection
      we are ready to transfect the 293T cell when it reaches ~80% confluency.
      1. prepare two 15ml tube, add 1ml OPTI-MEM to each tube.
      2. one tube add 40 ul lipofectamine 3000, the other tube add 40 ul P3000 reagent and the following plasmids:
      12ug pLOVE-Luciferase-EGFP + 6ug psPAX2 + 2ug S or S (1-1254aa) D614G, let stand for 5min
      3. transfer the mix in the first tube to the second tube, gently blow a few times, let stand for 15~20min
      4. remove Medium for 293T and add 3ml fresh Medium, then add the 2ml mixture dropwise to the medium
      5. replace with 10ml fresh Medium about 8h after transfection (about 6:00 PM)

  • 08/16/2020
    Sunday, August 16, 2020 6:00 PM

    We observe high transfection efficiency in 293T under fluorescence microscope 24h after transfection

  • 08/17/2020
    Monday, August 17, 2020 6:00 PM

    collect the suprenant 48h after transfection, and add 10ml fresh Medium

  • 08/18/2020
    Tuesday, August 18, 2020 6:00 PM

    • Collect and concentrate the viruses
      1. collect the suprenant 72h after transfection store at 4℃

  • 08/20/2020
    Thursday, August 20, 2020 6:00 PM

    • Collect and concentrate the viruses (continued)
      2. To remove cells in the collected suprenant, suprenant were centriguged by 900 rpm, 5min and then filtered through a 0.45 um filter.
      3. Pseudoviruses were concentrated by a centrifugal ultrafiltration device using BD tube ~22000g for 1h
      4. now we see virus clumps gathered at the bottom of the tube, remove the suprenant carefully and add 50ul Medium to dissolve virus.

  • 08/21/2020
    Friday, August 21, 2020 9:00 AM

    • Virus titer determination
      1. virus RNA extraction
      Following the manual of Viral RNA extraction Kit (TAKARA), we extracted the RNA of S and S-D614G pseudovirus
      the concentration of S and S-D614G viral RNA is 90ng/ul and 120ng/ul, respectly.

  • 08/22/2020
    Saturday, August 22, 2020 9:00 AM

    • Virus titer determination (continued)
      2. cDNA synthesis
      Following the manual of iScript cDNA Synthesis Kit (Bio-rad, 1708890), S and S-D614G cDNA were synthesis.
      5x iScript Reaction Mix 4ul
      iScript Reverse Transcriptase 1ul
      Nuclease-free water 3.9 ul for S, and 6.7 ul for S-D614G
      RNA template 1ug (S or S 1-1254aa)11.1ul for S, and 8.3 ul for S-D614G
      total 20ul

      incubate the complete reaction mix in a thermal cycler using the following protocol
      Priming 5 min at 25℃
      Reverse transcription 20 min at 46℃
      RT inactivation 1 min at 95℃
      Optional step Hold at 4℃

      the cDNA was stored at -80℃ when the reation is complete

  • 08/23/2020
    Sunday, August 23, 2020 9:00

    • Virus titer determination (continued)
      3. qPCR
      Following the manual of TransLvTM lentivirus qPCR Titration Kit (Transgen, FV201), we measured the titer of S and S-D614G.

      here’s the titer of S and S-D614G

      SS-D614G
      titer249080.15429638.838

  • 08/24/2020
    Monday, August 24, 2020 9:00 AM

    • Thaw cells
      thaw ACE2-293T cells (transfected with human ACE2) were purchased from ProCell.
      1. shaking the frozen ACE2-293T tube for ~60s
      2. transfer the ACE2-293T cell to 15ml tube and then add 3ml Medium, 900 rpm, centrifuge for 5 min
      3. remove supernatant, add 2ml Medium to resuspend ACE2-293T and count cells by automatic cell counter

  • 08/27/2020
    Thursday, August 27, 2020 9:00 AM

    ACE2-293T reachs ~90% confluency. cell was replate by 0.25% trpsin. 2x106 cells were plated into 100mm culture dish, we plate two, one is prepared for S, another for S-D614G

  • 08/29/2020
    Saturday, August 29, 2020 9:00 AM

    • Pseudovirus infection assay
      To compare the infection between S and S-D614G, we need to do infection assay by luciferase detection
      1. first, when ACE2-293T reachs ~90% confluency. cell was replate by 0.25% trpsin. 2x104 ACE2-293T cells were seeded into 96-well plates.
      2. 50 ul medium containing serial dilutions (1:1, 1:2, 1:4, 1:8, 1:16) of pseudoviruses (~ 6.4× 105 vg) was were added to the 96-well containing ACE2-293T cells.
      3. After 12 h of infection, 100ul fresh culture medium was added to each well.
      4. Luciferase activity was measured 48 h after infection

  • 08/30/2020
    Sunday, August 30, 2020 9:00 PM

    We observe high infection efficiency in ACE2-293T under fluorescence microscope 36h after adding pseudovirus

  • 08/31/2020
    Monday, August 31, 2020 9:00 PM

    • Pseudovirus infection assay (continue the step4: luciferase assay)
      4. Luciferase activity was measured 48 h after infection according to ONE-GloTM Luciferase Assay System Technical Manual (E6120, Promega).
      a. ONE-GloTM reagents were thawed at room temperature and mix well after thawing
      b. the 96-well containing ACE2-293T cells were took out from the incubator and equilibrated to room temperature before adding the reagent
      c. add a volumn of reagent equal to that of the culture medium in each well. Mix for optimal consistency. As we use 96-well plates, 100ul of reagent is added to the cells grown in 100ul of medium.
      d. wait for ~3 min to allow complete cell lysis and measure in a luminometer.
      SS-D614G
      4.00E+04684412610
      8.00E+042292728368
      1.60E+05132604164428
      3.20E+05495633597398
      6.40E+05616276938601

  • 09/03/2020
    Thursday, September 3, 2020 9:00 AM

    Next, we want to test whether this plateform can apply to evaluation of neutralizing antibody. We got serum sample, which is harvested at Day 35 post vaccination by an RBD vaccine, a kindly gift from Dr. Jingyun Yang (West China Hospital, Sichuan University), then we use this serum for neutralization assay.

    • Pseudovirus neutralization assay
      1. Firstly, we plate two 96-well with ACE2-293T, each well contains 2X104 cells. One plate for S, and another for S-D614G
      2. 12h after cell culture, 50 ul medium containing pseudoviruses (~4x104 vg) were incubated with media or with serially diluted sera from immunized with an RBD vaccine (from 1:1000 to 1:102400) for 1 h at 37℃.
      3. add the mix to the 96-well plates containing ACE2-293T cells.

  • 09/04/2020
    Friday, September 4, 2020 9:00 AM

    • Pseudovirus neutralization assay (continued)
      4. after 12 h of infection, 100ul fresh culture medium was added to each well.

  • 09/06/2020
    Sunday, September 6, 2020 9:00 AM

    • Pseudovirus neutralization assay (continued)
      Luciferase activity was measured 48 h after infection according to ONE-GloTM Luciferase Assay System Technical Manual as previously described.

      here’s the neutralization assay result

      dilution% neutralization% neutralization
      SD614G
      0.00292.966297.2637
      0.00190.581595.8541
      0.000575.16589.801
      0.0002564.51882.1725
      0.00012552.401675.2902
      0.000062544.548858.6235
      0.00003125 41.58634.4942
      0.00001562543.368538.0597
      0.000007812533.13135.9867
      0.0000039062538.59931.7579
      0.00000195312532.432431.675
Copyright by the team PYMS GZ China.