Cloning and Expression of TBEV NS4A in A549 cells

• Main Author: Gerdhem, Lovisa
• Format: Others
• Language: English
• Published: Örebro universitet, Institutionen för medicinska vetenskaper 2019
• Subjects: Medical and Health Sciences; Medicin och hälsovetenskap
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-78018

Background: Tick borne Encephalitis Virus (TBEV), the cause of the fatal Central Nervous System (CNS) infection Tick Borne Encephalitis (TBE), belongs to the family of Flaviviridae and the genusFlavivirus. In order to replicate themselves, they cause the formation of a replication complex, which is nestled inside vesicles of the Endoplasmic Reticulum (ER) membrane to protect the RNA from host defense mechanisms. These membrane vesicles are created by the interaction of viral nonstructural proteins and host proteins. The nonstructural protein, NS4A, is believed to play an important part in the formation of ER membrane vesicles, but it is not precisely known how. Earlier studies have shown that the cleavage of the NS4A protein from its C-terminal fragment,called 2k,is an important process in the formation of membrane vesicles. Aim: To express NS4Ain human A549 cells, and analyzeit with Western Blot in order to later purify said proteins and study their interactions with host proteins.This is important information in the study of mechanisms in membrane bending, which is crucial for the replication of TBEV as well as other flaviviruses. Methods: The DNA fragments were amplifiedusing PCR and tagged with a HIS-tag sequence. The fragments were then cloned into plasmids and transfected into A549 cells. SDS page and Western Blot were used to express the proteins. Results: The results showeda successfulexpression of the proteinsNS4A and NS4A-2k. The expression was stronger for the NS4A proteins lacking the 2k-fragment. The expression also seemed to be strongerin the cell cultures harvested on the second day of culturing. Conclusion: The results imply that the expression of NS4A is affected by whether or not it is cleaved from its 2k fragment. However,the experiment was only done once, and so,no absolute conclusions can be drawn.