Part:BBa_K2002000
LJM11 Saliva protein
LJM11 is a kratagonist. Serotonin binds to LJM11 via hydrogen bonds between its amino group and the side chains of Asn-342 and Thr-327 as well as with the carbonyl oxygen of Phe-344. LJM11 is found in the saliva of the sandfly Lutzomyia longipalpis, the vector for species of leishmaniasis such as Leishmania major and Leishmania infantum.
LJM11 is able to induce an immune response against the pathogen, but this ability is not linked to its serotonin-binding ability. Due to the positive charge on the surface of its top face, it is more effectively phagocytosed by macrophages and dendritic cells than if it were neutral or negatively charged. (Xu, Xueqing et al., 2011) Thus, an immune response can be mounted against it. Indeed, this protein has been shown to confer protective immunity against L. major transmitted by the sandfly in mouse experiments, conferring ulcer-free protection. This ability was first shown when administered without adjuvant by intradermal injection (Gomes, Regis et al.2012) It was then shown when a Listeria monocytogenes based vaccine which secreted this protein was administered by a number of parenteral routes, with the greatest protection being provided when administered intravenously. (Abi Abdallah, Delbert S. et al., 2014).
Assembly of construct:
The G-block with overlapping Gibson ends for the shuttle vector pNZ44 was ordered from IDT:
The overlapping regions were 28bp on the 5’ end of the G-block and 24bp on the 3’ end of the G-block. pNZ44 was digested with the single-cutting enzyme XbaI and purified using a ThermoScientific GeneJET PCR Purification Kit and its concentration determined using a nanodrop.
The genes were then assembled using NEB E5520S HiFi DNA Assembly Kit. An insert:plasmid ratio of 3:1 was used. The following construct was obtained:
After assembly, the construct was transformed into E.coli C2987I cells by the heat-shock method and 100μL was plated on two chloramphenicol LB agar plates, A and B. They were incubated overnight at 37℃. Six colonies were observed on plate A and one colony in plate B, and thus were labelled A1-6 and B1.
Colonies were screened using colony PCR. The primers used were: forward primer for USP45, reverse primer for pNZ44. The expected amplicon was ~300bp. PCR was carried out using 5x HOT FIREPol® Blend Master Mix Ready to Load. After PCR an agarose gel was run and the following was observed:
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Abi Abdallah, Delbert S. et al. “A Listeria Monocytogenes-Based Vaccine That Secretes Sand Fly Salivary Protein LJM11 Confers Long-Term Protection against Vector-Transmitted Leishmania Major.” Ed. J. L. Flynn. Infection and Immunity 82.7 (2014): 2736–2745. PMC. Web. 15 Oct. 2016.
Gomes, Regis et al. “Immunity to Sand Fly Salivary Protein LJM11 Modulates Host Response to Vector-Transmitted Leishmania Conferring Ulcer-Free Protection.” The Journal of Investigative Dermatology 132.12 (2012): 2735–2743. PMC. Web. 14 Oct. 2016.
Xu, Xueqing et al. “Structure and Function of a ‘Yellow’ Protein from Saliva of the Sand Fly Lutzomyia Longipalpis that Confers Protective Immunity against Leishmania Major Infection.” The Journal of Biological Chemistry 286.37 (2011): 32383–32393. PMC. Web. 15 Oct. 2016.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 509
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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