Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae

Abstract

Background: There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in E. coli, yeast and baculovirus grown in insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV).

Findings: Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in insect larvae. Recognition of G21-465 by ß-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (Oncorhynchus mykiss) was demonstrated.

Conclusions: Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.

Figure 1

Recognition of extract proteins from Trichoplusia ni insect larvae infected with recombinant baculoviruses BacNi (lanes A and E), G21-465 (lanes B and F), G21-507 (lanes C and G) and concentrated VHSV (lanes D and H) with anti polyHis (lanes A, B, C, D) or anti-gpG (lanes E, F, G, H) MAbs. Larvae were collected 72 hours after infection with the recombinant baculoviruses. Larvae were homogenized in guanidinium-free buffer and their soluble proteins were extracted. Thirty μg of protein extracts was loaded and electrophoresed in the same 12% polyacrylamide gel (PAGE) and transferred to a nitrocellulose membrane. The membrane was then cut and reacted with anti-His MAb (lanes A, B, C and D) or with anti-gpG MAb mix (lanes E, F, G and H) and peroxidase labelled anti-mouse Ig and then detected by chemiluminiscence. Some of the molecular weight markers on KDa are shown by the arrow numbers to the left and to the right.

Figure 2

Ni-affinity chromatography fractions of protein extracts from Trichoplusia ni insect larvae infected with recombinant baculoviruses, and PAGE of the pooled fractions of G21-465 (insert). Fifty recombinant baculovirus-infected larvae (~10 g) were homogenized in 6 M guanidinimun chloride, 1 M sodium chloride in 40 mM phosphate buffer at pH 7.8 containing 25 mM imidazole. They were then disrupted by sonication and centrifuged until a clear lysate was obtained. A 3-ml Probond (Invitrogen) bed column was used to retain the polyhistidine-tagged recombinant proteins. Bound proteins were eluted using the same buffer with 250 mM imidazole. ●, protein extract from larvae infected with the G21-465 recombinant baculovirus. ■, protein extract from larvae infected with the G21-507 recombinant baculovirus. *, protein extract from larvae infected with the BacNi baculovirus. Fractions with an absorbance > 0.3 at 280 nm were pooled and electrophoresed in a 4 to 20% polyacrylamide gel. Results of G21-465 stained with Coomassie-blue are shown in the insert.

Figure 3

Recognition of solid-phase G21-465 by anti-gpG MAbs and VHSV-hyperimmunized trout sera (hTS). Plates were coated with 1 μg per well of insect-derived G21-465 untreated (- ß) or treated with ß-mercaptoethanol (+ ß), as indicated in the text. Hyperimmunized trout sera (hTS) were obtained by intraperitoneal injections of purified VHSV and diluted 2000-fold. The MAb 2C9 was an anti-VHSV nucleocapsid N-MAb used as a negative control [29]. MAbs were used at 2.5 μg per well. Open bars, ELISA performed with dilution buffer adjusted to pH 6.7. Black bars, ELISA performed with dilution buffer adjusted to pH 7.7. Means and standard deviations from 4 experiments are shown.