Razvoj encimskoimunskega testa na osnovi nanotelesa SdAb19 za določitev koncentracije virusnega proteina Nef v plazmi

Mavec, Nina (author), Lenassi, Metka (mentor), Turk, Dušan (co-mentor)

Posamezniki z virusom HIV-1, ki se zdravijo s terapijo ART in imajo v krvi nezaznavno virusno obremenitev, vseeno nimajo povsem povrnjene imunske funkcije. Imajo tudi znatno večje tveganje za razvoj bolezni, povezanih z vnetjem, kot so rak, kardiovaskularne in nevrokognitivne bolezni. K pojavu omenjenih bolezni najverjetneje prispevajo tudi telesni rezervoarji virusa HIV-1, ki lahko proizvajajo nekatere virusne proteine in transkripte, kljub odsotnosti proizvodnje infektivnih virionov. Znano je, da se protein Nef virusa HIV-1 izloča iz okuženih celic z zunajceličnimi vezikli in da je prisoten v krvni plazmi polovice aviremičnih posameznikov, zato je dober kandidatni označevalec za ovrednotenje aktivnih telesnih rezervoarjev virusa HIV-1. Trenutno obstaja le en komercialni encimskoimunski test za detekcijo proteina Nef, ki pa ima številne pomanjkljivosti. Z uporabo nanotelesa SdAb19, ki ima široko specifičnost za protein Nef različnih sevov virusa HIV-1, hkrati pa protein veže z nanomolarno afiniteto, bi rešili nekatere od teh pomanjkljivosti. Hipoteza magistrskega dela je, da bomo z izražanjem heksahistidinsko označenih nanoteles v E. coli in čiščenjem izraženih nanoteles z nikljevo afinitetno kromatografijo in kromatografijo z ločevanjem po velikosti pridobili zadostne količine nanotelesa SdAb19 za uporabo v encimskoimunskem testu za detekcijo virusnega proteina Nef. Za kasnejšo biotinilacijo in imobilizacijo nanotelesa SdAb19 na podlago pri encimskoimunskem testu smo izrazili tudi bakterijsko biotin ligazo BirA, označeno s proteinom, ki veže maltozo. Z optimizacijo pogojev izražanja smo določili najuspešnejše pogoje za izražanje nanoteles v bakterijah. Uspešno je bilo izražanje v citoplazmi celic E. coli BL21[DE3], za pridobivanje nanoteles v topni obliki pa je bilo pomembno, da je izražanje potekalo počasi, kar smo dosegli z gojenjem kultur pri nizki temperaturi, s stresanjem pri nizkih vrtljajih ter z uporabo laktoze kot induktorja. V bakterijah smo uspešno izrazili nanotelesa SdAb19 in SdAb19-AviTag in jih očistili z nikljevo afinitetno kromatografijo in kromatografijo z ločevanjem po velikosti. Pri enakih pogojih smo izrazili in očistili tudi bakterijsko biotin ligazo BirA-MBP. V nadaljevanju bomo nanotelesa modificirali, da jih bomo lahko uporabili za lovljenje ali detekcijo proteina Nef v encimskoimunskem testu.

• Language: Slovenian
• Keywords: Nef, encimskoimunski test, nanotelo, SdAb19, izražanje
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-121601
• COBISS.SI-ID: 38566915
• Publication date in RUL: 19.10.2020

Secondary language

• Language: English
• Title: Nanobody-based immunoassay for the detection of viral protein Nef in plasma

Abstract

HIV-1 infected individuals on antiretroviral therapy with an undetectable viral load do not have completely restored immune function and have a greater risk for developing inflammatory diseases such as cancer, cardiovascular disease and neurocognitive diseases. HIV reservoirs, capable of producing viral proteins and transcripts even in the absence of mature virion production, likely contribute to the development of these diseases. HIV-1 viral protein Nef was shown to induce its own release from infected cells in extracellular vesicles and was detected in the plasma of half of HIV-infected aviremic individuals. It is therefore a good candidate biomarker for the evaluation of active HIV-1 reservoir. Currently, only one commercial immunoassay for Nef detection is available, which has many drawbacks. By using nanobody SdAb19, which shows a broad specificity for Nef from different HIV-1 strains and a nanomolar affinity for Nef binding, we can address some issues and improve the performance of the immunoassay. The hypothesis of this work is to produce sufficient amounts of SdAb19 for detection of viral protein Nef in the immunoassay by expressing His-tagged nanobodies in E. coli and purifying them with nickel affinity chromatography and size exclusion chromatography. We have also produced the bacterial biotin ligase BirA, tagged with maltose binding protein, which will be used to biotinylate nanobody SdAb19-AviTag to ensure immobilization of the nanobody in the immunoassay. By performing optimization of expression, we determined the optimal parameters for expression of SdAb19 in bacteria. Expression in the cytoplasm of E. coli strain BL21[DE3] was most successful, while it was crucial that the rate of expression was slow in order to produce nanobodies in soluble form. Slow rate of expression was ensured by growing cultures at low temperature, shaking at low speed and using lactose for induction. We successfully expressed nanobodies SdAb19 and SdAb19-AviTag, which we purified by using nickel affinity chromatography and size exclusion chromatography. Additionaly, we expressed and purified the bacterial biotin ligase BirA-MBP. Moving forward, we will modify the produced nanobodies and use them in the immunoassay for the capture or detection of Nef.

• Keywords: Nef, immunoassay, nanobody, SdAb19, expression