The uncleaved signal peptide of Bombyx mori nucleopolyhedrovirus GP64 preferentially activates BmSpz7 expression, contributing to protein secretion.

Bombyx mori nucleopolyhedrovirus (BmNPV) significantly impacts silkworm sericulture, causing substantial economic losses. The GP64 protein, a primary envelope protein of BmNPV budded virus (BV), retains its signal peptide (SP) in the mature form, crucial for its translocation to the plasma membrane (PM) and viral infectivity. This study investigates the role of the uncleaved SP of GP64 in activating the expression of BmSpz7, a novel Spätzle family member identified through RNA-seq analysis. We cloned and characterized BmSpz7, demonstrating its upregulated expression in BmN cells and silkworm larvae infected with BmNPV containing GP64 with an uncleaved SP. Additionally, transient expression of GP64's SP significantly enhanced BmSpz7 expression and protein secretion. These findings suggest that the uncleaved SP of GP64 plays a pivotal role in activating BmSpz7, providing new insights into the molecular interactions between BmNPV and its host, and revealing potential targets for antiviral strategies in sericulture.

Bombyx mori Nucleopolyhedrovirus Hijacks Multivesicular Body as an Alternative Envelopment Platform for Budded Virus Egress.

Baculovirus budded virus (BV) acquires its envelope and viral membrane fusion proteins from the plasma membrane (PM) of the host cell during the budding process. However, this classical BV egress pathway has been questioned because an intracellularly localized membrane fusion protein, SPΔnGP64 (glycoprotein 64 [GP64] lacking the signal peptide [SP] n region), was assembled into the envelope to generate infective BVs in our recent studies. Here, we identify an additional pathway for Bombyx mori nucleopolyhedrovirus (BmNPV) BV assembly and release that differs, in part, from the currently accepted model for the egress pathway of baculovirus. Electron microscopy showed that during infection, BmNPV-infected cells contained many newly formed multivesicular body (MVB)-like compartments that included mature virions at 30 h postinfection (p.i.). Immunoelectron microscopy demonstrated that the MVBs contained CD63, an MVB endosome marker, and GP64, a BmNPV fusion glycoprotein. MVB fusion with the PM and the release of mature virions, together with naked nucleocapsids, were observed at the cell surface. Furthermore, MVB egress mediated the translocation of SPΔnGP64 to the PM, which induced cell-cell fusion until 36 h p.i. This BV egress pathway can be partially inhibited by U18666A incubation and RNA interference targeting MVB biogenesis genes. Our findings indicate that BmNPV BVs are enveloped and released through MVBs via the cellular exosomal pathway, which is a subordinate BV egress pathway that produces virions with relatively inferior infectivity. This scenario has significant implications for the elucidation of the BmNPV BV envelopment pathway. IMPORTANCE BmNPV is a severe pathogen that infects mainly Bombyx mori, a domesticated insect of economic importance, and accounts for approximately 15% of economic losses in sericulture. BV production plays a key role in systemic BmNPV infection of larvae. Despite the progress made in the functional gene studies of BmNPV, BmNPV BV egress is ill-understood. This study reports a previously unreported MVB envelopment pathway in BmNPV BV egress. To our knowledge, this is the first report of a baculovirus using dual BV egress pathways. This specific BV egress mechanism explains the cause of the non-PM-localized SPΔnGP64-rescued gp64-null bacmid infectivity, elucidating the reason underlying the retention of SP by BmNPV GP64. The data obtained elucidate an alternate molecular mechanism of baculovirus BV egress.

Label-free proteomics analysis on the envelope of budded viruses of Bombyx mori nucleopolyhedrovirus harboring differential localized GP64.

Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 is the key membrane fusion protein that mediates budded virus (BV) infection. We recently reported that BmNPV GP64's n-region of signal peptide (SP) blocked the SP-cleavage and mediated GP64 localization on the plasma membrane (PM); n-region (SP∆nGP64) absence caused GP64 intracellular localization, however, SP∆nGP64 was still incorporated into virion to generate BVs with lower infectivity. To better understand the biogenesis of the envelope of BmNPV BV, we conducted a label-free ESI mass spectrometry analysis of the envelope of purified BVs harboring PM localized GP64 or intracellular localized SP∆nGP64. The results indicated that 31 viral proteins were identified on the envelope, among which 15 were reported in other viruses. The other 16 proteins were first reported in BmNPV BV, including the BmNPV-specific protein BRO-A and proteins associated with vesicle transportation. Six proteins with significant intensity differences were detected in virions with differential localized GP64, and five specific proteins were identified in virions with GP64. Meanwhile, we identified 81 host proteins on the envelope, and seven lipoproteins were first identified in baculovirus virion; other 74 proteins are involved in the cytoskeleton, DNA-binding, vesicle transport, etc. In the meantime, eight and five specific host proteins were, respectively, identified in GP64 and SP∆nGP64's virions. The two virions shared 68 common host proteins, and 8 proteins were identified on their envelopes with a significant difference. This study provides new insight into the protein composition of BmNPV BV and a clue for further investigation of the budding mechanism of BmNPV.

Environmental polycyclic aromatic hydrocarbon exposure is associated with low back pain.

Several studies have assessed the influence of several often-ignored environmental factors on low back pain (LBP), but the effects of environmental polycyclic aromatic hydrocarbon (PAH) exposure on LBP are unclear. During the 2001-2004 cycle of the National Health and Nutrition Examination Survey (NHANES), our study was given to a representative sample of US participants older than 20 (N = 2743). Environmental PAH exposure was calculated using urinary PAH metabolite concentrations. Weighted logistic regression was performed to assess the connection between PAH levels and LBP, with mediation analysis utilised to explore the underlying mechanism. Levels of 1-hydroxynaphthalene (1-OHNa), 2-hydroxynaphthalene (2-OHNa) and total PAHs had a statistically significant positive association with LBP. The odds ratios per 1-unit increase for log-transformed levels of urinary 1-OHNa, 2-OHNa, and total PAHs with LBP were 1.01 (95% CI 1.02-1.19), 1.19 (95% CI 1.04-1.36) and 1.16 (95% CI 1.03-1.32), respectively. The results revealed a strong dose-response association between 1-OHNa, 2-OHNa, total PAHs, and LBP risk. Subgroup analysis indicated that 2&3-OHPh may increase the risk of LBP in the lower family income subgroup. Gamma-glutamyl transaminase (GGT), known as a biomarker of oxidative stress, was strongly related to PAHs. The relationship between total PAHs and LBP was mediated in part by GGT. Our study demonstrates associations between environmental PAH exposure and LBP that need more research to determine the precise effects of various PAH compounds on LBP.

The cytoplasmic tail substitution increases the assembly efficiency of Ebola virus glycoprotein on the budded virus of Bombyx mori nucleopolyhedrovirus.

Glycoprotein (GP1,2) of the Ebola virus (EBOV) is the key membrane fusion protein, which is a key candidate protein for vaccine preparations. Previously, GP1,2 was expressed by Bombyx mori nucleopolyhedrovirus (BmNPV) expression vector system; however, few GP1,2 was incorporated into budded virus (BV) of BmNPV. To improve the incorporation efficiency of GP1,2 into the virion, the GP1,2 fusion with the cytoplasmic tail of GP64 of BmNPV was expressed in BmN cells by the BmNPV expression system. The BV was purified by ultracentrifugation, and GP1,2 expression in BV was detected by the antibody. The result indicated that a 532% increase in the relative GP1,2 densitometry signal was observed in constructs utilizing the GP64 C-terminal domain; moreover, the substitution of GP1,2 native signal peptide with GP64 signal peptide increased the incorporation efficiency by 34.6% in the relative GP1,2 densitometry signal. We revealed that the application of the cytoplasmic tail of BmNPV GP64 significantly increased the incorporation rate of GP1,2 into the BV envelope. This study lays a foundation for GP1,2 vaccine development.

Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses.

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Identification of endoplasmic-reticulum-associated proteins involved in Bombyx mori nucleopolyhedrovirus entry by RNA-seq analysis.

Membrane fusion is a key step in enveloped virus infection, releasing the viral genome into the cytoplasm to initiate infection. Bombyx mori nucleopolyhedrovirus (BmNPV) is an enveloped DNA virus that mainly infects silkworms. Information about membrane fusion of BmNPV with host cells is still limited. In this study, BmN cells were pretreated with ??ammonium chloride??, and infection with BmNPV was allowed to occur naturally through endocytosis or artificially through low-pH-induced fusion with the plasma membrane, after which the cells were subjected to RNA-seq. The results indicated that a few endoplasmic reticulum-associated proteins (ERAPs) were among the common upregulated DEGs, including BiP, CRT, and HSP90, and this upregulation was confirmed by q-PCR. Knockdown of BiP, CRT, and HSP90 expression by siRNA resulted in significant inhibition of BmNPV infection. This study suggests that ERAPs may be involved in the BmNPV membrane fusion process during infection.

Involvement of the neddylation modification system in Bombyx mori nucleopolyhedrovirus replication.

Neddylation is a posttranslational modification that is similar to ubiquitination, and involved in some critical biological processes, such as DNA repair, transcription regulation, and ubiquitin-proteasome pathway. Recently, it was found that neddylation inhibitor MLN4924 has potent antiviral activity against human viruses including herpes simplex virus (HSV)-1, HSV-2, and influenza viruses. Here, we report that MLN4924 could dramatically and dose-dependently inhibits the propagation, formation of budding virus (BV) and occlusion body (OB) of a lepidopteran virus-Bombyx mori nucleopolyhedrovirus (BmNPV), impaired OB assembly. In addition, the neddylation modification protein NEDD8 is colocalized with aggresome and autophagosome. Our findings suggest that inhibiting neddylation could be an antibaculovirus strategy and MLN4924 may be used as candidate drug for that purpose.

18 Additional Amino Acids of the Signal Peptide of the Bombyx mori Nucleopolyhedrovirus GP64 Activates Immunoglobulin Binding Protein (BiP) Expression by RNA-seq Analysis.

GP64 is the key membrane fusion protein of Group I baculovirus, and while the Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 contains a longer n-region (18 amino acid) of the signal peptide than does the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the function of the n-region has not been determined. In this study, we first showed that n-region is required for membrane protein localization in BmN cells, then the transcriptome sequencing was conducted on proteins guided by different signal peptide regions, and the results were analyzed and validated by quantitative PCR and luciferase assays. The results indicated that 1049 differentially expressed genes (DEGs) were identified among the different region of signal peptides and the control. With the n-region, the protein export pathway was upregulated significantly, the Wnt-1 signaling pathway was downregulated, and BiP was significantly activated by the GP64 full-length signal peptide. Furthermore, RNA interference on BiP efficiently increased luciferase secretion. These results indicate that the GP64 n-region plays a key role in protein expression and regulation.

Infectious hematopoietic necrosis virus truncated G protein effect on survival, immune response, and disease resistance in rainbow trout.

The major antigenic protein of infectious hematopoietic necrosis virus (IHNV) is the surface glycoprotein G, which contains neutralizing epitopes that induce the production of immune neutralizing antibodies. In this study, the IHNV G gene sequence was truncated according to bioinformatics principles and then recombinantly expressed via an E. coli expression system. We then assessed the specific antibody immunoglobin M (IgM) levels of rainbow trout immunized with recombinant truncated G protein (emulsified with Freund's incomplete adjuvant), and showed that antibody IgM levels of immunized fish were significantly higher than in the control group (p < 0.01). The mRNA expression levels of interferon 1 (IFN1) and interleukin-8 (IL-8) were also up-regulated significantly (p < 0.01) in head kidneys and spleens of rainbow trout immunized with recombinant truncated G protein. Also, after challenge with wild-type IHNV HLJ-09 virus on Day 28, rainbow trout immunized with recombinant truncated G protein showed cumulative survival rates of 60%. These results indicate that the truncated G protein of IHNV expressed by the E. coli prokaryotic expression system can be used as a candidate immunogen for an IHNV subunit vaccine, which lays a theoretical foundation for the study of further potential IHNV subunit vaccines.

The role of infectious hematopoietic necrosis virus (IHNV) proteins in recruiting the ESCRT pathway through three ways in the host cells of fish during IHNV budding.

In cytokinetic abscission, phagophore formation, and enveloped virus budding are mediated by the endosomal sorting complex required for transport (ESCRT). Many retroviruses and RNA viruses encode "late-domain" motifs that can interact with the components of the ESCRT pathway to mediate the viral assembly and budding. However, the rhabdovirus in fish has been rarely investigated. In this study, inhibition the protein expression of the ESCRT components reduces the extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in IHNV release. The respective interactions of IHNV proteins including M, G, L protein with Nedd4, Tsg101, and Alix suggest the underlying molecular mechanism by which IHNV gets access to the ESCRT pathway. These results are the first observation that rhabdovirus in fish gains access to the ESCRT pathway through three ways of interactions between viral proteins and host proteins. In addition, the results show that IHNV is released from host cells through the ESCRT pathway. Taken together, our study provides a theoretical basis for studying the budding mechanism of IHNV.

An amino acid duplication/insertion in the Bm126 gene of Bombyx mori nucleopolyhedrovirus alters viral gene expression as shown by differential gene expression analysis.

Open reading frame (ORF) 126 (Bm126) of Bombyx mori nucleopolyhedrovirus (BmNPV) is not essential for viral replication, and two subtypes of this gene have been identified in China. The Bm126-SX subtype encodes a protein with a simple amino acid duplication/insertion relative to the Bm126-GD subtype; however, significant differences in the cytopathic effect and infectivity of viruses carrying these variant genes have been observed. To elucidate the cause of these differences, differential gene expression analysis was performed at the early stage of infection with viruses harbouring variants of Bm126. Differential expression was observed for 103, 209, and 313 host genes and 9, 44, and 67 viral genes in vGD126 samples relative to the control samples (vSX126) at 6, 12, and 24 h postinfection, respectively. These results indicated that the duplication/insertion in Bm126 altered the viral expression pattern. The differentially expressed host genes were found to be related to ribosome, spliceosome, and proteasome pathways, and several factors involved in signal transduction were also identified. The differential expression of these viral and host genes was confirmed by qPCR. This study indicates that the amino acid duplication/insertion in the Bm126 gene has a biological function related to the regulation of viral gene expression and serves as a basis for further characterization of Bm126 gene function.

Preincubation with a low concentration of methyl-ß-cyclodextrin enhances baculovirus expression system productivity.

OBJECTIVES: To enhance the productivity of foreign protein in culture cells using baculovirus expression system. RESULTS: A low concentration of MßCD, with the optimal application concentration of 0.25 mM and the appropriate preincubation time range from 10 to 120 min, can efficiently enhance expression levels in both the AcMNPV and BmNPV expression systems. CONCLUSIONS: Preincubation with a low concentration MßCD enhance baculovirus infection and foreign protein expression productivity.

Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.

Enhanced Parkinson's gait, reduced fall risk, and improved cognitive function through multimodal rehabilitation combined with rivastigmine treatment.

OBJECTIVE: This study aimed to examine the effects of combined rehabilitation and rivastigmine treatment on patients with Parkinson's disease (PD). METHODS: Gait parameters were assessed using the Gibbon Gait Analyzer in fifteen patients. Baseline gait data and cognitive assessments were collected. Each patient underwent external counterpulsation therapy, transcranial magnetic stimulation therapy, and exercise therapy for one hour per day, five days a week for three weeks. Post-intervention, gait and cognitive data were re-evaluated. Alongside their standard PD medications, all participants were administered rivastigmine throughout the study period. RESULTS: The intervention significantly enhanced motor function in the single-task test, evidenced by marked improvements in gait metrics such as stride width and walking speed, and a substantial reduction in fall risk. Cognitive function, assessed by mini-mental state examination and Montreal cognitive assessment, showed an improvement trend after the three-week intervention. Improvements in dual-task walking function were observed, although these changes did not reach statistical significance. CONCLUSION: Multimodal exercise training combined with rivastigmine treatment significantly improves certain gait parameters in the single-task test, enhances balance, and reduces the risk of falling in patients with PD. Cognitive function also demonstrated improvement.

Intracellular localized heterogeneous protein franking by a transmembrane domain of GP64 is sufficient to be assembled on budded virions of Bombyx mori nucleopolyhedrovirus.

Baculovirus has been widely used for foreign protein expression in biomedical studies, and budded virus (BV) surface display has developed into an important research tool for heterogenous membrane protein studies. The basic strategy of surface display is to construct a recombinant virus where the target gene is fused with a complete or partial gp64 gene. In this study, we further investigate and develop this BV surface displaying strategy. We constructed stable insect cell lines to express the target protein flanking with different regions of signal peptide (SP) and GP64 transmembrane domain (TMD). Subsequently, recombinant BmNPV was used to infect the cell, and the integration of heterogeneous protein into BV was detected. The results indicated that deletion of the n-region of SP (SPΔn) decreased the incorporation rate more than that of the full-length SP. However, the incorporation rate of the protein fused with h and c-region deletion of SP (SPΔh-c) was significantly enhanced by 35-40 times compare to full-length SP. Moreover, the foreign protein without SP and TMD failed to display on the BV, while the integration of foreign proteins with GP64 TMD fusion at the c-terminal was significantly enhanced by 12-26 times compared to the control. Thus, these new strategies developed the BV surface display system further.

Unveiling non-classical glycosylation patterns in Bombyx mori nucleopolyhedrovirus GP64: Insights into viral entry and fusion.

The glycoprotein GP64 of alphabaculovirus is crucial for viral entry and fusion. Here, we investigated the N-glycosylation patterns of Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 and its signal peptide (SP) cleaved form, SPΔnGP64, along with their impacts on viral infectivity and fusogenicity. Through deglycosylation assays, we confirmed N-glycosylation of BmNPV GP64 on multiple sites. Mutational analysis targeting predicted N-glycosylation sites revealed diverse effects on viral infectivity and cell fusion. Particularly noteworthy were mutations at sites 175, which resulted in complete loss of infectivity and fusion capacity. Furthermore, LC-MS/MS analysis uncovered unexpected non-classical N-glycosylation sites, including N252, N302, N367, and N471, with only N302 and N471 identified in SPΔnGP64. Subsequent investigation highlighted the critical roles of these residues in BmNPV amplification and fusion, underscoring the essentiality of N367 glycosylation for GP64 fusogenicity. Our findings provide valuable insights into the non-classical glycosylation landscape of BmNPV GP64 and its functional significance in viral biology.

The impact of sarcopenia on the incidence of postoperative outcomes following spine surgery: Systematic review and meta-analysis.

PURPOSE: Sarcopenia is considered to be an important predictor of adverse outcomes following spinal surgery, but the specific relationship between the two is not clear. The purpose of this meta-analysis is to systematically review all relevant studies to evaluate the impact of sarcopenia on spinal surgery outcomes. METHODS: We systematically searched PubMed, Embase and the Cochrane Library for relevant articles published on or before January 9, 2023. The pooled odds ratio (OR) with 95% confidence intervals (CIs) was calculated in a random effects meta-analysis. The main outcome was the risk of adverse outcomes after spinal surgery, including adverse events and mortality. This systematic review and meta-analysis was conducted following the PRISMA guidelines to evaluate the impact of sarcopenia on spinal surgery outcomes. In addition, we also conducted a subgroup analysis and leave-one-out sensitivity analyses to explore the main sources of heterogeneity and the stability of the results. RESULTS: Twenty-four cohort studies, with a total of 243,453 participants, met the inclusion criteria. The meta-analysis showed that sarcopenia was significantly associated with adverse events (OR 1.63, 95% CI 1.17-2.27, P < 0.001) but was no significantly associated with mortality (OR 1.17, 95% CI 0.93-1.46, P = 0.180), infection (OR 2.24, 95% CI 0.95-5.26, P < 0.001), 30-day reoperation (OR 1.47, 95% CI 0.92-2.36, P = 0.413), deep vein thrombosis (OR 1.78, 95% CI 0.69-4.61, P = 0.234), postoperative home discharge (OR 0.60, 95% CI 0.26-1.37, P = 0.002) and blood transfusion (OR 3.28, 95% CI 0.74-14.64, P = 0.015). CONCLUSION: The current meta-analysis showed that patients with sarcopenia have an increased risk of adverse events and mortality after spinal surgery. However, these results must be carefully interpreted because the number of studies included is small and the studies are significantly different. These findings may help to increase the clinicians' awareness of the risks concerning patients with sarcopenia to improve their prognosis.

Risk factors of postoperative delirium following spine surgery: A meta-analysis of 50 cohort studies with 1.1 million participants.

Objectives: Postoperative delirium (POD) is considered to be a common complication of spine surgery. Although many studies have reported the risk factors associated with POD, the results remain unclear. Therefore, we performed a meta-analysis to identify risk factors for POD among patients following spinal surgery. Methods: We systematically searched the PubMed, Embase and the Cochrane Library for relevant articles published from 2006 to February 1, 2023 that reported risk factors associated with the incidence of POD among patients undergoing spinal surgery. The Meta-Analysis of Observational Studies in Epidemiology (MOOSE) guidelines were followed, and random effects models were used to estimate pooled odds ratio (OR) estimates with 95 % confidence intervals (CIs) for each factor. The evidence from observational studies was classified according to Egger's P value, total sample size, and heterogeneity between studies. Results: Of 11,329 citations screened, 50 cohort studies involving 1,182,719 participants met the inclusion criteria. High-quality evidence indicated that POD was associated with hypertension, diabetes mellitus, cardiovascular disease, pulmonary disease, older age (>65 years), patients experiencing substance use disorder (take drug ≥1 month), cerebrovascular disease, kidney disease, neurological disorder, parkinsonism, cervical surgery, surgical site infection, postoperative fever, postoperative urinary tract infection, and admission to the intensive care unit (ICU). Moderate-quality evidence indicated that POD was associated with depression, American Society of Anesthesiologists (ASA) fitness grade (>II), blood transfusion, abnormal potassium, electrolyte disorder, length of stay, inability to ambulate and intravenous fluid volume. Conclusions: Conspicuous risk factors for POD were mainly patient- and surgery-related. These findings help clinicians identify high-risk patients with POD following spinal surgery and recognize the importance of early intervention.

[Construction of recombinant lentiviral vector containing integrin ß1 shRNA and its effects on integrin ß1 gene expression in Sombati cell culture model of refractory epilepsy].

OBJECTIVE: To construct a recombinant lentiviral vector containing integrin ß1 shRNA to provide an effective tool for integrin ß1 gene effect and a possible mechanism of Sombati cell of clinical refractory epilepsy. METHODS: Four lentiviral vectors containing integrin ß1 shRNA were constructed and transfected into 293T cells. PC12 cells were infected by concentrated lentivirus and the gene-silencing efficiency was verified. And the most effective lentivirus containing shRNA was selected with Western blot. Then neonatal rat hippocampal neurons and Sombati cells were infected by lentivirus containing shRNA and the gene-silencing efficiency was also monitored by Western blot. RESULTS: RNAi lentivirus expression vectors targeting rat integrin ß1 gene were successfully constructed and confirmed by DNA sequencing. The recombinant lentivirus particles were packaged successfully to produce a sufficient titer for subsequent experiments. The expression of protein significantly decreased in rat hippocampal neurons and rat Sombati cells after vector transfection. CONCLUSION: The recombinant lentiviral vector containing integrin ß1 shRNA is constructed successfully. And the gene-silencing effects are effective and stable in neonatal rat hippocampal neurons and Sombati cells.