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Objective:To construct a recombinant herpes simplex virus 2 (HSV-2) expressing enhanced green fluorescent protein (EGFP) using clustered, regularly interspaced, short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology.Methods:Four strategies for inserting exogenous EGFP gene into HSV-2 genome using CRISPR/Cas9 technology were designed: (1) conventional homology-directed repair: circular two homology arm donor-mediated gene knock-in; (2) linearized single homology arm donor-mediated gene knock-in; (3) homology-independent targeted integration; (4) conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.Results:The recombinant virus HSV-2-EGFP was successfully constructed based on the second, the third and the fourth strategies. The second strategy was the most efficient, followed by the third and the fourth strategies. The purified recombinant virus could stably express green fluorescent protein in seven passages and shared similar growth characteristics in Vero cells to the parental virus.Conclusions:Linearized single homology arm donor could increase the efficiency of gene knock-in, and cell lines stably expressing Cas9 and sgRNA could increase the efficiency of gene knock-in mediated by homology-directed repair.
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Objective:To investigate the effects of genomic location of a foreign gene in Shanghai-191 strain measles virus (MV) vector on gene expression and virus replication.Methods:The nucleotide sequence encoding S1 protein of SARS-CoV-2 was inserted at different positions in MV antigenome (the upstream of the N gene, between P and M genes, between H and L genes), and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and N, P, and L proteins, respectively. The transfected cells were lysed and the supernatants were used to infected Vero cells to harvest recombinant viruses. S1 proteins expressed by the recombinant viruses were identified by RT-PCR, indirect immunofluorescence assay, Western blot and ELISA. Growth kinetics of the recombinant viruses were analyzed.Results:Recombinant viruses were failed to be rescued when the S1 protein-coding sequence was cloned into the upstream of N gene. Two recombinant viruses, MV-M-S1 and MV-L-S1, were successfully rescued when cloning the S1 protein-coding sequence into the intergenic region between P and M genes, or H and L genes, and could express S1 protein. MV-M-S1 expressed more S1 protein than MV-L-S1, but the titer of MV-M-S1 was lower.Conclusions:Inserting a foreign gene at different positions in the MV genome might have different effects on gene expression and virus replication. This study provided reference for the subsequent construction of MV vector.
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Herpes simplex virus (HSV), the main pathogen of human herpetic diseases, is an attractive viral vector due to a wide range of host cell types and a large capacity of foreign gene. The manipulation on herpes simplex virus genome, up to 152 kb, has been the key process and has undergone the improvement from homologous recombination to bacterial artificial chromosome (BAC) and CRISPR/Cas9. However, homologous recombination is imperfect because the efficiency of recombination is relatively low and the product is too complex to purify. Construction of recombinant HSV based on BAC is time-and labor-consuming. Currently, CRISPR/Cas9 has been applied to genetic engineering of various species by more and more researchers due to its convenience, good repeatability, low cost, and then the manipulation of viral genome is more accurate and efficient. Application of CRISPR/Cas9 in the research of HSV gene function, HSV infection therapy and viral vector were summarized in this review, in order to provide reference for corresponding research.
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Objective@#To develop a real-time quantitative PCR (qPCR) assay for detecting Sendai virus gene copy number.@*Methods@#The recombinant plasmid was constructed and a reference used to establish the real-time qPCR method with the TaqMan probe and verified for reproducibility and sensitivity.@*Results@#The linear range of the developed real-time qPCR assay was 1.024 × 103 ~5 × 1010 copies/μl, with an R2 value of more than 0. 99. The standard recovery of the tested samples was 84.56%~119.48%.@*Conclusions@#Compared with traditional hernagglutination assay for particle number detection, this method has higher sensitivity, better repeatability and high quantitative accuracy. It can be used for the detection of particle number of vaccine with sendai virus as the carrier, so as to detect the specific activity of vaccine products.
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This study was aimed to compare the mechanical characteristics under different physiological load conditions with three-dimensional finite element model of rigid fixation and elastic fixation in the lumbar. We observed the stress distribution characteristics of a sample of healthy male volunteer modeling under vertical, flexion and extension torque situation. The outcomes showed that there existed 4-6 times pressure on the connecting rod of rigid fixation compared with the elastic fixations under different loads, and the stress peak and area of force on elastic fixation were much higher than that of the rigid fixations. The elastic fixation has more biomechanical advantages than rigid fixation in promoting interbody lumbar fusion after surgery.
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Humans , Male , Biomechanical Phenomena , Finite Element Analysis , Lumbar Vertebrae , General Surgery , Models, Theoretical , Pressure , Range of Motion, Articular , Spinal FusionABSTRACT
Objective To analyze the genome sequencing of mumps virus strain 79 purified from working seeds plague and examine its phylogenetic relationship with wild type virus strain isolated in China,and to explore the efficacy of vaccine at molecular level.Methods Whole genome sequences of two substrains of S79(major and minor),were obtained with fragment amplification by RT-PCR.Genetic distances between S79 and strains identified in China were analyzed based on the phylogenic analysis of small hydrophobic protein(SH) sequences.Results The nucleotide homologies of two S79 substrains(major and minor),with a ratio of 2:5 during culture,with Jeryl Lynn(JL) strain were 99.7% and 100%,respectively.There were scattered non-homologous recombination between two substrains.The genetic distances between strain S79,which was genotype A,and wild type virus strain identified in China,which were genotype F,was 11.2% -20.0%.S79 live vaccine was composed of two substrains,major and minor component,which were highly similar to JL strain in their genome sequences,but different from JL in their ratios during culture.Conclusion Different from wild type virus strain identified in China,the genotype of S79 was A,and phylogenetically distant from other strains,which may account for the low efficacy of S79 live vaccine.The ratios of two substrains might also be of interest for further study of the vaccine protection and efficacy.
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Objective Through smdying the distribution of the Sappan acetic ether extract in transplanted heart of rats,to discuss selectivity of sappan in donor's hearts.Methods Improved Ono model was used to replicate the heart transplantation model of rats.Successfully operated rats were separated into 2 groups:a model group and a sappan group.Distributions of sappan in transplanted heart were observed by HPLC-FPC.Results One kind of original substance.of sappan was detected in transplanted heart sample,but not in un-transplanted hearts.Conclusion The substance of sappan may have selectivity in transplanted heart.