Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely
distributed in Africa, Asia, Russia and the Balkans. The causative agent, CCHF
virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality
rate. Cases of CCHF are diagnosed annually in southern Africa. Increasing
numbers of cases are seen in regions of Asia and in the past ten years CCHFV has
emerged in several countries in the Balkans and re-emergence in south-western
regions of the Russian Federation. Diagnosis of CCHFV infections during the acute
phase is based on isolation of the virus or amplification of viral RNA. Patients that
survive the infection have a demonstrable IgG and IgM antibody response, usually
from day 5 to 7 after onset of illness. Current serological diagnostic assays based
on ELISA or IF use inactivated virus which requires biosafety level 4 facilities for
culturing the virus and therefore limits the number of laboratories that can prepare
suitable reagents. Preparation of recombinant antigens would enable laboratories to
perform serological diagnosis of CCHFV infections and surveillance studies. The
purpose of this study was to prepare a recombinant CCHFV nucleoprotein using a
bacterial expression system, to determine if the protein was immunogenic and to
determine if the protein was able to detect IgG antibodies in survivors of CCHFV
infection.
The complete open reading frame of the gene encoding the NP of CCHFV was
amplified by RT-PCR using primers specifically designed with restriction sites
engineered to the primers to facilitate cloning. The amplicon was cloned into
pGEM® T Easy vector using T/A cloning and the gene sequenced to confirm that
the correct gene had been amplified and cloned into the vector for downstream
cloning and expression applications. Initially we aimed to express the native gene
using a bacterial expression system and the NP gene was rescued from the
recombinant plasmid and cloned into pQE-80L vector using the BamH1 and Pst1
restriction sites present in the multiple cloning site on the vector. Various attempts
were made to express the CCHFV NP protein however no protein was detectable
using SDS PAGE methods or Western blot. The nucleotide sequence that we had determined for the open reading frame of our
gene encoding the NP was analysed using the Rare Codon Analysis Tool software
and we elected to codon optimize the gene for expression in E. coli. The optimized
gene was synthesized by GenScript and supplied cloned in the multiple cloning site
of pUC57. The optimized gene was excised from pUC57 and cloned into pColdTF
bacterial expression vector. A 106 kDa protein was expressed from the construct
likely representing the HIS tagged TF chaperone protein fused to the CCHFV NP
protein and confirmed by Western blot analysis. A higher yield of the protein was
present in the insoluble phase and as optimization of the growth and induction
conditions did not significantly alter the insoluble to soluble ratio of the expressed
protein, the protein was harvested from the insoluble phase by denaturing,
purification and refolding of the protein. The biological activity of the recombinant
protein was confirmed using immunoassays and by immunizing mice to determine if
the antibodies induced by the recombinant protein could be detected using an
antigen prepared from the whole virus. Four of five mice immunized with the
recombinant NP had a detectable antibody response using an immunofluorescent
assay. Serum samples from acute and convalescent patients collected at varying
stages after onset of illness were reacted in a Western blot with the recombinant
CCHFV NP protein. The recombinant antigen was able to detect IgG antibody in all
the convalescent patient sera except two sera collected on days 14 and 15 during
the acute phase. In contrast all the samples were detected using the recombinant
antigen in an ELISA. Due to the potential biohazardous nature of samples only
samples collected two weeks after onset of illness were tested. The results showed
100% concordance with the results obtained in an ELISA using mouse brain
derived antigen. The assay was shown to be reproducible and stability studies
showed that four months after preparation the protein was still active. A full
validation of the protein using a large panel of serum samples from confirmed
CCHF patients is now required.
The results suggest that bacterially expressed proteins lacking post translational
modifications and folding that occur with mammalian and baculovirus expression
can be used in ELISA to detect IgG antibody against CCHFV in human sera which
finds application in diagnostics, epidemiologic and surveillance studies.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-10042011-095133 |
| Date | 04 October 2011 |
| Creators | Samudzi, Rudo Ruth |
| Contributors | Prof FJ Burt |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-10042011-095133/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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