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用于基因疫苗的巨噬细胞相关载体的比较分析

Comparative analysis of macrophage associated vectors for use in genetic vaccine.

作者信息

Ahsan Mohammad Feraz, Gore Milind M

机构信息

National Institute of Virology, Pashan Campus, 130/1, Sus Road, Pashan, Pune, 411021, India.

出版信息

Genet Vaccines Ther. 2011 Jun 18;9(1):10. doi: 10.1186/1479-0556-9-10.

DOI:10.1186/1479-0556-9-10
PMID:21682913
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3146807/
Abstract

BACKGROUND

Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used.

METHODS

Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries.

RESULTS

All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines.

CONCLUSIONS

Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.

摘要

背景

非专职抗原呈递细胞（APC）呈递抗原可导致无反应性。在基因疫苗中，靶向巨噬细胞和APC以实现高效抗原呈递可能会导致平衡的免疫反应。一种这样的方法是在要使用的载体中掺入APC特异性启动子。

方法

选择三个已知在巨噬细胞中具有活性的启动子，并克隆到哺乳动物表达载体（pAcGFP1-N1）中，分别用巨唾液酸蛋白、EmrI和β-5整合素启动子构建（pAcGFP-MS）、（pAcGFP-EMR）和（pAcGFP-B5I）。使用CMV启动子的（pAcGFP-CMV）作为阳性对照，无启动子载体（pAcGFP-NIX）作为阴性对照。在每个启动子的控制下，使用绿色荧光蛋白（GFP）基因作为读出指标。使用流式细胞术和带有TaqMan探针化学法的定量逆转录-聚合酶链反应（qRT-PCR）分析巨噬细胞和非巨噬细胞系中GFP的表达。

结果

通过荧光、蛋白质印迹和定量逆转录-聚合酶链反应观察到，所有相关启动子在巨噬细胞系细胞中均占主导地位。巨唾液酸蛋白的活性显著高于其他巨噬细胞启动子。CMV启动子在巨噬细胞系中的活性高1.83倍。与非巨噬细胞系相比，巨唾液酸蛋白启动子驱动的GFP在巨噬细胞衍生细胞系中24小时后的表达高4.40倍。

结论

基于本研究，巨唾液酸蛋白启动子可用于靶向巨噬细胞优势表达。其作为候选疫苗的效用需要进行体内研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/f8cb13f08136/1479-0556-9-10-8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/0a7ee681cf4b/1479-0556-9-10-1.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/aedbac6cea26/1479-0556-9-10-6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/09665768dd42/1479-0556-9-10-7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/f8cb13f08136/1479-0556-9-10-8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/0a7ee681cf4b/1479-0556-9-10-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/43a39ae37323/1479-0556-9-10-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/33dfdc5827db/1479-0556-9-10-3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/58faf8d97173/1479-0556-9-10-4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/c2ac5f147f23/1479-0556-9-10-5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/aedbac6cea26/1479-0556-9-10-6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/09665768dd42/1479-0556-9-10-7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1589/3146807/f8cb13f08136/1479-0556-9-10-8.jpg

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