PCDH-His-LprA-FLAG慢病毒载体在293T细胞中的过表达分析

Mocking of p CDH-His-LprA-FLAG Lentiviral Vector of Mycobacterium Tuberculosis Lipoprotein A and Overexpression of the Lentivirus in 293T Cells

构建PCDH-His-LprA-FLAG慢病毒表达载体,同时分析其在293T细胞中的过表达。将结核分枝杆菌标准菌株H37Rv的DNA当成分析模板,对结核分枝杆菌脂蛋白A(LprA)基因实施扩展处理。通过连接慢病毒载体pCDH-CMV-MCS-EF1-Puro的T4酶,塑造pCDH-LprA。采用PCR测序完成阳性克隆载体的检测;采用重组质粒以及协同质粒融合转染293T细胞包装病毒,获取慢病毒颗粒上清液,通过梯度稀释法完成病毒滴度的检测,采用Western blot对病毒在293T细胞基因的表达实施检测。成功塑造PCDH-His-LprA-FLAG慢病毒表达载体。病毒侵染的293T细胞内PVRL-2的表达量提升了100倍,PPARa的表达量提升4. 5倍。蛋白结构预测说明Necitn-2是包含信号肽的跨膜蛋白。PPARa表达量提升导致结核分枝杆菌相关基因的活化,提升机体免疫力,信号肽和跨膜结构说明LprA蛋白会同膜中蛋白受体融合,驱动下游脂代谢的信号通路。Mtb细菌的LprA脂蛋白能够控制巨噬细胞、促进细胞增长和吞噬能力。通常情况下,慢性病毒可以进行细胞侵染的种类较多,且慢病毒可以将表达载体整合到宿主细胞的基因组上,实现在宿主细胞上连续的目的基因表达。慢性病毒具有的这种特点被广泛应用于细胞体外实验中。因此将含有毒性的Mtb因子当成治疗结核病的疫苗,为结核病的治疗提供了可靠的分析依据,将LprA当成预防以及治疗结核病的药物靶点。

To establish the expression vector of PCDH-His-lpra-flag slow virus,and to analyze its expression in 293 T cells,the DNA of Mycobacterium tuberculosis H37 Rv was used as an analytical template for the extended treatment of the lipoprotein A( Lpr A)gene of Mycobacterium tuberculosis. PCDH-LprA was engineered by T4 enzyme ligating the lentiviral vector p CDH-CMV-MCS-EF1-Puro. The positive cloning vector was detected by PCR sequencing. The 293 T cell packaging virus was transfected with recombinant plasmid and co-plasmid,and the lentiviral particle supernatant was obtained. The virus titer was detected by gradient dilution method. Western blot was used to detect the gene expression in 293 T cells. PCDH-His-LprA-FLAG lentiviral expression vector was successfully constructed. The expression of PVRL-2 in 293 T cells infected by virus increased by 100 times,and the expression of PPARa increased by 4. 5 times. Protein structure prediction indicates that Necitn-2 is a transmembrane protein containing signal peptide. Increased expression of PPARa leads to the activation of genes associated with Mycobacterium tuberculosis. Signal peptide and transmembrane structure indicate the fusion of protein receptors in Lpr A membrane and drive the signaling pathways of lipid metabolism downstream. The Lpr A lipoprotein of Mtb bacteria controls macrophages,and promotes cell growth and phagocytosis. In general,chronic viruses can carry out a variety of cell infections,and lentivirus can integrate the expression vector into the genome of the host cell to achieve continuous expression of the target gene on the host cell. This feature of chronic viruses is widely used in cell in vitro experiments. Therefore,the use of toxic Mtb factor as a vaccine for tuberculosis provides a reliable basis for the treatment of tuberculosis,using Lpr A as a drug target for the prevention and treatment of tuberculosis.