Overview
实验设计主要是介绍巴拉巴拉巴拉巴拉（总览这一板块的内容）
Plasmid Preparation and Validation
To investigate the role of Enterolobium Contortisiliquum Trypsin Inhibitor (EcTI) in inhibiting the trypsin activity in the termite killing process, the EcTI-His tag sequence was cloned into certain plasmid backbones with J23119 promoter and Ampicillin resistance gene to harvest EcTI. The plasmid was custom-synthesized by VectorBuilder (a commercial gene synthesis and plasmid construction service) and extracted using a standard plasmid isolation kit (Medium-Speed Plasmid Extraction Kit (Universal Type), Beyotime D0020). It was quantified via UV spectrophotometry (dsDNA setting) to ensure suitability for downstream experiments.
• J23119-EcTI-His tag (wild type EcTI with flag tag for expression validation): 207.3 ng/μL
Plasmid Construction and Expression Validation
To provide an oxidative environment more conducive to the formation of disulfide bonds of EcTI, we chose to express the constructed plasmid in the Shuffle T7 strain. For complex proteins that require the formation of disulfide bonds (such as enzymes or secreted proteins containing multiple cysteines), the environment in Shuffle T7 can significantly reduce misfolded folding and increase the yield of active proteins. While J23119 acts as a strong constitutive promoter, its continuous and efficient drive of gene expression may lead to excessive protein accumulation and increased folding pressure. Shuffle T7 precisely alleviates this problem.
EcTI Function Analysis
After obtaining EcTI from the Shuffle T7 strain, a trypsin activity assay using the Colorimetric Trypsin Activity Assay Kit (Beyotime P0324S) was conducted to analyze the inhibition effect of our product. The kit involves using a substrate labeled with pNA). Trypsin hydrolyzes this substrate to release free pNA, whose concentration can be detected by measuring absorbance at a specific wavelength. The change in pNA concentration reflects trypsin activity, and by comparing pNA concentration changes, the inhibition effect of EcTI on trypsin activity can be assessed.
3.1 Preparation of bacterial supernatant
• Control: Shuffle T7 strain without plasmid
• EcTI: Shuffle T7 carrying the J23119-EcTI-His tag plasmid
The bacterial culture was collected after overnight incubation and then centrifuged and washed with PBS buffer to remove LB Broth. Subsequently, the bacteria were subjected to ultrasonic lysis.
3.2 Result analysis
As shown in Fig.1a, the pNA concentration change of the control group (Control (D10)) rose gradually over time, reaching nearly 6 nmol at 60 minutes. In contrast, the pNA concentration changes of different dilution EcTI treatment groups (EcTI (D10), EcTI (D1000), EcTI (D100000)) remained almost stable and were much lower than that of the control group. Fig.1b demonstrated that the trypsin activity of the control group (Control (D10)) was about 6 nmol/hour, while the trypsin activities of different dilution EcTI treatment groups were significantly lower than that of the control group, and the differences among these EcTI treatment groups were relatively small, which proved EcTI could effectively inhibit trypsin activity.
Figure 1.a: 图片说明.
Figure 1.b: 图片说明.
To assess EcTI's inhibitory effect on trypsin activity, one-way ANOVA and Tukey's multiple comparisons were performed. ANOVA showed a significant overall group difference (F = 6.460, P = 0.0049), and the Brown-Forsythe test confirmed homogeneous variances (P = 0.2474). Tukey's test revealed that compared to the Control Plasmid group, remaining EcTI-treated groups (D10, D1000, and D100000) had significantly lower trypsin activity (P < 0.05 for D1000 and D100000; P < 0.01 for D10), indicating potent inhibition. Even at a high dilution (D100000), EcTI was effective. However, no significant differences existed between remaining EcTI-treated groups (P > 0.05), suggesting no clear concentration-dependent gradient in inhibition.
References
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