Objective To optimize the method of transcription activator‐like effector transcription factors (TALE‐TFs) con‐struction ,some improvement and adaption were made based on the traditional methods .Methods We first constructed the basic tandem fragments with different length ,including trimer ,tetramer ,pentamer and hexamer by Golden Gate cloning technique and PCR ,then the procedure with the highest efficacy was chosen to construct our TALE‐TFs .To determine the function of the TALE‐TFs ,the plasmid pminCMV with the specific binding sequence of TALE‐TFs was constructed by fragment substitution reaction (FSR) .The transcription activating function of TALE‐TFs was confirmed by the intensity of red fluorescence ,after TALE‐TFs , pEGFP‐N1 and pminCMV plasmid were co‐transfected into 293HEK cells .Results An optimized method for TALE‐TFs construc‐tion and functional assay was established .Conclusion This method can potentially be wildly used in fields that the expression of some constitutively expressed genes needs to be modified .

Objective To evaluate the real-time genotyping and quantitative PCR(RT-GQ-PCR)method by comparing it with direct sequence analysis and the multiplex-PCR method.Methods RT-GQ-PCR,direct sequence analysis and the multiplex-PCR method were used to detect HBV genotypes of 113 patient samples with HBV-DNA positive.ResultsThe detection rate of RT-GQ-PCR and direct sequence analysis was 100%,and the multiplex-PCR is 94.69%.The concordance between RT-GQ-PCR and the multiplex-PCR is perfect(Kappa value =0.915),and the consistency of RT-GQ-PCR and direct sequence analysis is pretty good(Kappa value = 0.742),specially at detecting single genotype.Twenty-eight samples with genotypes B and C dual infections were detected by RT-GQ-PCR,but only 19 samples by the multiplexPCR and 13 samples by direct sequence analysis.Conclusion The RT-GQ-PCR is convenient,rapid and accurate in HBV genotyping,especially more sensitive than direct sequence analysis and the multiplex-PCR for detecting dual genotypes.The method is applicable for large-scale epidemiological study.

Hepatitis B virus (HBV) DNA replication takes place in the viral capsid that consists of 180 or 240 copies of HBV capsid (HBc or core) protein. The monomeric core protein contains an apical loop region that forms the spikes on the surface of viral capsid upon core dimerization and capsid assembly. To investigate the impact on HBV DNA replication through gene engineering at the spike of HBV capsid. plasmids expressing engineered HBc with linker-fused enhanced green fluorescent protein (EGFP) or shortened EGFP insertion at the spike region were constructed by Restriction Digestion and Ligation-independent Cloning (RLIC). The wildtype or mutant HBc construct was cotransfected with HBV1.1c(-), a plasmid containing 1.1 unit-length HBV genome with deficiency in HBc expression, into HEK293 cells, respectively. GFP signal was observed through a fluorescence microscope and HBV DNA replicative intermediates were assayed by Southern blotting to determine the expression and functions of different recombinants. Our results demonstrated that the RLIC method was effective to generate deletion or insertion in the apical loop region of HBc. Both HBc-EGFP recombinants with different linkers produced green fluorescence but with different subcellular distribution pattern. However, HBV DNA replication was not detected with the trans-complementation of these two HBc recombinants. In addition, other recombinants including the one only with the deletion of aa79-80 failed to support HBV replication. Taken together, our results suggest that RLIC is a robust method which can be broadly applied in gene engineering; different peptide linkers may have different influences on the functions of an engineered fusion protein; and HBc aa79-80 play a critical role for HBc to support HBV DNA replication.
Capsid Proteins ; genetics ; Cloning, Molecular ; Genetic Engineering ; methods ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; physiology ; Humans ; Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection ; Virus Replication

Objective To investigate the effect of fine nursing on the rehabilitation of patients with severe traumatic brain injury after large bone flap craniotomy.Methods A total of 80 severe craniocerebral injury patients were randomly divided into observation group and control group,with 40 cases per group.Patients of two groups were treated with large bone flap craniotomy treatment in perioperation,the observation group was treated with refined care,and control group received traditional nursing.Psychology,cognitive function and motor function recovery and complications on awaken two postoperative days after anesthesia (T1),discharge day (T2) and 6 months after discharge (T3) were recorded and compared.Results At T2 and T3,observation group had lower SAS score and SDS,MMSE score and NCSE score than control group (P ＜ 0.05).Observation group had lower ADL score,FMA score and incidence of complications than control group (P ＜ 0.05).Conclusion Refined nursing can significantly improve psychology,cognitive and physiological rehabilitation of patients with severe craniocerebral injury in the frontal and temporal lobe,and effectively prevent the complications.

Objective To investigate the effect of fine nursing on the rehabilitation of patients with severe traumatic brain injury after large bone flap craniotomy.Methods A total of 80 severe craniocerebral injury patients were randomly divided into observation group and control group,with 40 cases per group.Patients of two groups were treated with large bone flap craniotomy treatment in perioperation,the observation group was treated with refined care,and control group received traditional nursing.Psychology,cognitive function and motor function recovery and complications on awaken two postoperative days after anesthesia (T1),discharge day (T2) and 6 months after discharge (T3) were recorded and compared.Results At T2 and T3,observation group had lower SAS score and SDS,MMSE score and NCSE score than control group (P ＜ 0.05).Observation group had lower ADL score,FMA score and incidence of complications than control group (P ＜ 0.05).Conclusion Refined nursing can significantly improve psychology,cognitive and physiological rehabilitation of patients with severe craniocerebral injury in the frontal and temporal lobe,and effectively prevent the complications.

The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Genetic Vectors ; genetics ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Membrane Proteins ; biosynthesis ; genetics ; physiology ; Mice ; Mice, Transgenic ; Microfilament Proteins ; genetics ; Muscle Proteins ; genetics ; Mutant Proteins ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

The persistent infection of hepatitis B virus (HBV) is the result of lacking specific immunity against the virus. This state is also called immune tolerance to HBV. In most cases, acute HBV infection in adults can induce specific immune response which can clear the virus. Perinatal HBV infection, however, usually progresses to chronic infection, indicating a defect in HBV-specific immune response. A typical specific immune response includes four processes, which were antigen presentation, specific CD4 + T cell activation, specific CD8 + T cell activation and B cell activation. There must be some dysfunctions in some or all of the four processes during chronic HBV infection. This article discussed the relationship between chronic HBV infection and cellular immunity, hoping to provide a reference for further study on the reconstitution of specific immunity against HBV.