前文细菌分泌系统总览梳理了细菌的几大分泌系统，现对其用于表达分泌蛋白，做几个案例解析：
1. 改造成团泛属菌Pantoea agglomerans表达分泌抗疟蛋白
文献：Wang, S., Ghosh, A.K., Bongio, N., Stebbings, K.A., Lampe, D.J., and Jacobs-Lorena, M. (2012) Fighting malaria with engineered symbiotic bacteria from vector mosquitoes. Proc Natl Acad Sci U S A 109:12734-12739.
We used the E. coli hemolysin (Hly)A system (20–22) to promote protein secretion from P. agglomerans.
利用的是I类分泌系统：
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I类分泌系统概述：
This type 1 secretion system requires three components to form a membrane pore (inner membrane components HlyB and HlyD and outer membrane component TolC). The HlyB–HlyD complex recognizes the C-terminal secretion signal of HlyA to guide direct export of HlyA-fusion proteins from the bacterial cytoplasm into the extracellular environment bypassing the periplasmic space.
I型分泌系统(type I secretion system，T1SS) 广泛存在于革兰氏阴性细菌中，该系统结构简单且依赖于含ATP结合盒的输出体。其主要有 HlyB、HlyD 和TolC 这3 种关键蛋白，这3种蛋白具有一定的协同作用，缺少任何一个蛋白都无法完成蛋白的胞外分泌[26]。大多数情况下，该系统分泌的蛋白通过识别C 端存在的 46−60 个氨基酸残基的信号肽，以未折叠的状态通过一个由内膜ATP 结合盒转运体、膜融合蛋白和外膜孔TolC组成的跨膜通道，实现底物蛋白的胞外运输，在胞外进行正确的折叠[27]，形成具有一定结构的蛋白，如图1 所示。
该系统主要分泌多糖、脂蛋白、维生素B12、金属螯合物 和多种小肽信号分子等。
应用载体：
效应分子表达载体(融合Effector protein and HlyA)：pEHlyA2SD
辅助质粒(HlyB and HlyD)：pVDL9.3 (Plasmid #168299)
The TolC protein of P. agglomerans is conserved and did not have to be supplied
质粒图谱：
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原文作者及原始文献或者addgene皆没有对应的序列，笔者根据已有的信息，根据文献使用引物，合理逆推，用snapgene拼接出质粒图谱，如下图：
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文中这一处，添加pnptII，用到XbaI和EcoRI衔接不上，推测是文章描述错误
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质粒出处：
- Fernández, L.A., Sola, I., Enjuanes, L., and de Lorenzo, V. (2000) Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of the hemolysin system. Appl Environ Microbiol 66:5024-5029. https://doi.org/10.1128/aem.66.11.5024-5029.2000
- Tzschaschel, B.D., Guzmán, C.A., Timmis, K.N., and de Lorenzo, V. (1996) An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nat Biotechnol 14:765-769. https://doi.org/10.1038/nbt0696-765
2. 改造粘质沙雷菌(serratia marcescens)表达分泌抗疟蛋白
文献：Wang, S., Dos-Santos, A.L.A., Huang, W., Liu, K.C., Oshaghi, M.A., Wei, G., Agre, P., and Jacobs-Lorena, M. (2017) Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria. Science 357:1399-1402.
We used the Serratia HasA (heme-binding protein) exporting system (13) (fig. S10A) to test five potent anti-Plasmodium effector molecules(11).
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Figure: The HasA secretion system consists of three components (HasD, HasE and HasF) that form a membrane pore and the HasA C-terminal secretion signal domain, to guide the fusion protein through the pore and out of the bacterial cell.
应用质粒载体：
表达荧光质粒（转座子）：pBAM2-GFP and pBAM2-mCherry
表达效应蛋白质粒：PDB47-mPLA2-HlyA,将HlyA替换为HasA.
载体出处：
- Létoffé, S., Ghigo, J.M., and Wandersman, C. (1994) Secretion of the Serratia marcescens HasA protein by an ABC transporter. J Bacteriol 176:5372-5377.
属实没有看明白
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