Expression, Purification Of VP1Fusion Protein In Escherichia Coli Of CVA16Virus And Research The Detection Methods

Coxsackie virus (Coxsackievirus, CV) can cause respiratory infections, HFMD, febrilerash, herpes angina, and myocarditis, acute flaccid paralysis and encephalitis and otherdiseases, A group of16type Coxsackie VP1protein (CVA16VP1) is a major pathogenmain treatment of intestinal virus. The VP1protein of the gene region encoding most ofCVA16viral epitopes and determine viral antigenicity.The research of CVA16is stillweak today, so the study aims to get VP1protein prokaryotic gene expression and proteinpurification through and the initial establishment of CVA16-IgM capture enzymeimmunoassay method, preparation and evaluation methods as vaccine reagents.The experimental arrangement and design phases have done a lot of work. First,according to the laboratory saved CVA16VP1, VP2and VP3sequencing report coloniescontaining the full-length sequence, using the primer5.0software design VP1codingregion downstream primer from CVA16cultures were amplified VP1gene sequence of theprotein, recombinant plasmid pET-41a (+)/VP1; secondly, to determine colony PCRpositive clones colonies and digested initially identified as positive recombinant plasmidpET-41a (+)/VP1transformed into E. coli BL21-Gold (DE3) pLysS, sequenced correctlyinduced, purified by SDS-PAGE analysis of protein expression, the conditions to optimizethe induction and expression products were purified if the results are consistent, andWestern Blot identification of immunoreactive; Finally, by recombinant VP1proteinpurified horseradish peroxidase labeled, the checkerboard titration to determine the initialconcentration and the coating concentration of enzyme conjugate, the initial capture ELISAfor detection.After a series of experiments above, we get some results: First, CVA16VP1gene wassuccessfully amplified fragment and the recombinant plasmid pET-41a (+)/VP1;secondly,we built a highly expressed CVA16prokaryotic expression strain VP1protein anddetermined he optimal conditions for inducible expression; SDS-PAGE showed that therecombinant protein and was consistent with the expected inclusion expression; purified by affinity chromatography and ion exchange chromatography and obtained a purified proteinconcentration analysis by Adobe photoshop cs6purity of recombinant proteins derived;weconfirmed by Western Blot detection of recombinant protein VP1reactions can occur withthe corresponding antibody which is good antigenicity. At the end of this experiment, weconfirmed that the best anti-μ antibody concentration package and enzyme labeled antigenthe best dilution, initially established a capture ELISA for detection CVA-IgM. The methodspecificity, sensitivity is better than other conditions, this conclusion can be used for theearly diagnosis of Coxsackie virus infection.