Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection.

Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus.

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION: A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC.

OBJECTIVE: In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. METHODS: A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. RESULTS: Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. CONCLUSION: An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

TALENs-mediated homozygous CCR5Δ32 mutations endow CD4+ U87 cells with resistance against HIV­1 infection.

Since evidence suggests that transplantation of bone marrow stem cells with the C­C chemokine receptor type 5 (CCR5)Δ32/Δ32 genotype may cure patients infected with human immunodeficiency virus (HIV)­1, the present study aimed to reproduce the CCR5Δ32 mutation in cluster of differentiation (CD)4+ U87 cells using genome engineering methods. A modified transcription activator­like effector nucleases (TALENs) technique, combined with homologous recombination for site­specific, size­controlled and homozygous DNA deletions, was used to reproduce the homozygous CCR5Δ32 mutation in CD4+ U87 cells. The results indicated that the frequency of the TALENs­targeted mutation reached 50.4% without any selection, whereas homologous recombination from CCR5 to CCR5Δ32 occurred in 8.8% of targeted cells. Notably, a HIV­1 challenge test demonstrated that CCR5Δ32/Δ32 CD4+ U87 cells were resistant to HIV infection. In conclusion, engineered CCR5Δ32/Δ32 mutations endowed CD4+ U87 cells with resistance against HIV­1 infection; this site­specific, size­controlled and homozygous DNA deletion technique was able to induce precise genomic editing, i.e., the deletion or insertion of a predetermined length of DNA sequence at a specific locus throughout the genome.

[Inhibition of HIV-1 in vitro by combination of vpr and tat specific short hairpin RNA via lentiviral vectors].

Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.

cDNA, genomic sequence cloning and overexpression of glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) from the Giant Panda.

GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence of some diseases. The cDNA and the genomic sequence of GAPDH were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mu musculus, Rattus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could be used for further functional studies.

cDNA cloning and overexpression of ribosomal protein S19 gene (RPS19) from the Giant Panda.

RPS19 is a component of the 40S small ribosomal subunit encoded by RPS19 gene. The cDNA of RPS19 was cloned successfully for the first time from the Giant Panda using RT-PCR technology. It was also sequenced, analyzed preliminarily, and expressed in Escherichia coli. The length of cDNA fragment cloned is 469 bp, and it contains an open-reading frame of 438 bp encoding 145 amino acids. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence share a high homology with those of Homo sapiens, Mus musculus, and Rattus norvegicus by 95.89%, 92.47%, and 93.61%, and 100.00%, 99.31%, and 99.31%, respectively. Topology prediction shows that there is one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, one tyrosine kinase phosphorylation site, three N-myristoylation sites, one amidation site, and one ribosomal protein S19e signature in the RPS19 protein of the Giant Panda (Ailuropoda melanoleuca). The RPS19 gene was overexpressed in E. coli and expression confirmed by western blotting. The results indicated that the RPS19 gene can be readily expressed in E. coli, and the N-terminally GST-tagged protein was a 42 kDa polypeptide, in good agreement with the predicted molecular weight. The protein obtained could be purified and its function studied.

cDNA, genomic sequence cloning and overexpression of ribosomal protein S25 gene (RPS25) from the Giant Panda.

RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a "living fossil", are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first report on the RPS25 gene from the Giant Panda. The data will enrich and supplement the information about RPS25, which will contribute to the protection for gene resources and the discussion of the genetic polymorphism.

cDNA, genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda.

AlphaB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further.

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda.

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.