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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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Laboratory test is the routine method of diagnosis, monitoring and blood screening of HIV infection, and main basis for early diagnosis of AIDS. HIV is divided into HIV-1 and HIV-2 subtypes, HIV-1 infection is the major cause of AIDS pandemic, while HIV-2 infection occurs in limited areas in the world, mainly in West Africa. HIV-2 infection has been reported in China since 1998. They are sporadic cases, and mainly HIV-1/HIV-2 mixed infections. There are less concerns about HIV-2 detection in China at present, and domestic HIV-2 detection reagents have not come into the market. At present, the detection method of HIV-2 is mainly antibody test and nucleic acid test. The initial screening is through rapid test and other methods and the confirmation is depended on Western Blot and Line Immune Assay. According to the HIV antibody test results, HIV-2 infection is confirmed. With the rapid development of molecular biology, the diagnostic method of nucleic acid detection laboratory has made great progress.
Résumé
Objective To explore the clinical effect of creatine phosphate on infantile with rotaviral enteritis and myocardial damage.Methods One hundred and twenty children with rotaviral enteritis and myocardial damage were randomly divided into the control group (n =60) and observation group (n =60).Two groups were given antiviral,rehydration,nutritional support,redressing acid-base imbalance and electrolyte disorders and other conventional treatment.The patients of the control group were given 160 mg/(kg · d) 1,6Fructose Diphosphate(1 times/d) on the basis of routine treatment,while those in the observation group were given 1.0 g creatine phosphate + 150 ml 5％ glucose injection(1 times/d) on the basis of routine treatment,both courses of two groups were 7 days.The levels of myocardial enzymes were measured and the clinical effect and adverse reactions were recorded.Results The total effective rate in observation group was 95.0％ (57/60),obviously higher than that of control group (80.0％ (48/60)) and the differences were statistically significant (x2 =6.268,P ＜ 0.05).There were no statistical significant difference in terms of myocardial enzyme between the two groups before treatment(P ＞0.05).The level of lactate dehydmgenase(LDH),creatine kinase (CK),creatine kinase isoenzyme (CK-MB) and aspartate aminotransferase (AST) of observation group were (163.8 ±18.5) U/L,(152.8±26.4) U/L,(19.4±7.8) U/Land (24.4 ±6.8) U/Lafter treatment,and (193.4 ± 22.8) U/L,(168.6 ± 32.6) U/L,(27.4 ± 9.2) U/L and (35.4 ± 9.8) U/L of control group,and the differences between the two groups were significant (t =3.484,3.228,4.278 and 4.729; P ＜ 0.05).There was no obvious adverse reaction during the treatment course.Conclusion The clinical effect of creatine phosphate on infantile with rotaviral enteritis and myocardial damage is remarkable and safe,and it is worth of clinical application.