title: Expression of a truncated hepatitis E virus capsid protein in the protozoan organism Leishmania tarentolae and its application in a serological assay.
abstract: Zoonotic infections with hepatitis E virus (HEV) genotype 3 are presumably transmitted via contaminated pig meat products, which raises the necessity for enhanced serological surveillance of pig herds. The aim of the study was to set up a novel protein expression system to overcome the well-known problems in (HEV-) protein expression using the standard Escherichia coli tools such as inclusion body formation and loss of protein conformation. A recombinant strain of the protozoan organism Leishmania tarentolae (L. tarentolae) was therefore established. A fragment of HEV ORF2 coding for a truncated capsid protein of a porcine HEV strain was cloned and parts of the plasmid DNA were introduced into the Leishmania genome, resulting in stably transformed cells. Via a C-terminal His-tag the recombinant HEVΔORF2 protein could be purified and concentrated directly from the medium, resulting in a total protein amount of approximately 1.4 mg/l Leishmania culture. The recombinant protein was coated on ELISA plates and was proven to be highly reactive and well-suited to be applied in a serological assay. By investigating 144 porcine sera, the in-house assay detected specific antibodies in 43.1% of the samples and demonstrated a higher sensitivity than a commercially available antibody test. Taken together, it was shown that L. tarentolae exhibits a remarkable alternative expression strategy for viral antigens with considerable advantages of a eukaryotic protein expression host.
authors.affiliation: Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Germany. christine.baechlein@tiho-hannover.de
authors.forename: C
authors.initials: C
authors.lastname: Baechlein
authors.forename: D
authors.initials: D
authors.lastname: Meemken
authors.forename: G
authors.initials: G
authors.lastname: Pezzoni
authors.forename: C
authors.initials: C
authors.lastname: Engemann
authors.forename: B
authors.initials: B
authors.lastname: Grummer
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression; D042822:Genomic Instability; D006508:Hepatitis Antibodies / Q000097:blood*; D016751:Hepatitis E / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D016752:Hepatitis E virus / Q000235:genetics / Q000276:immunology*; D007891:Leishmania / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology; D014730:Veterinary Medicine / Q000379:methods; D014773:Virology / Q000379:methods
chemical_list: D036022:Capsid Proteins; D006508:Hepatitis Antibodies; D011994:Recombinant Proteins
keywords: Antigen expression; Leishmania tarentolae; Pig; Serology; Zoonosis