Recombinant sugar binding proteins and their functional analysis: Mannan-binding protein

 The human MBP cDNA is excised from the vector by digestion with SmaI and SacI and is subcloned into a vaccinia virus transfer vector, pBSF2-16.

 In the resultant transfer vector (pBSF2-16/MBP), the human MBP cDNA is located immediately downstream of the A-type inclusion body of a cowpox virus (ATI) hybrid promoter, and the human MBP cDNA flanked by the ATI hybrid promoter is interposed in the hemagglutinin (HA) gene, a selection marker for obtaining recombinant vaccinia viruses.

 5 μg of pBSF2-16/MBP and 10 mg of intact genomic DNA extracted from wild-type WR vaccinia virus are diluted in 1.5 mL of OptiMEM, and 30 μg of a cationic liposomal transfection reagent is diluted in 1.5 mL of OptiMEM. These two solutions are then mixed gently and incubated at room temperature for 45 min to yield the DNA-liposome complex.

 The cultured COS-7 cells (2.5 × 10⁵ cells in a 10-cm dish) are infected with 2.5 × 10⁴ PFU of IBT-dependent vaccinia virus for 1 h. The cells are exposed to the transfection solution for 8 h at 37˚C under 5% CO₂.

 After 20 h incubation, the cells are harvested, and then the virus progeny is released by sonication. Monolayer culture of RK13 cells are infected with the progeny virus.

 The virus progeny obtained from HA-negative plaques inoculated onto monolayer culture of RK13 cells and further screened for the expression of human MBP by immunohistochemical staining with a monoclonal antibody specific for human MBP.

 The virus progeny obtained from MBP-positive plaqures is selected once more to purify the recombinant vaccinia virus.

 HLF cells at a density of 1 × 10⁷ cells per 75 cm² are infected with the recombinant vaccinia virus containing the human MBP cDNA at a multiplicity of infection (MOI) of 5, and then the infected cells are incubated for 48 h at 37˚C under 5% CO₂.

 The culture supernatant of HLF cells is harvested and centrifuged to remove cell debris and viral particles.

 The resulting supernatant is mixed with a half volume 3 × loading buffer, filtered through a 0.45 μm filter, and then applied to a mannan-Sepharose 4B column that has been equilibrated with loading buffer.

 After washing the column with loading buffer, the bound recombinant proteins are eluted with elution buffer.

 The eluted fractions are pooled and concentrated. The purity of the recombinant MBP can be checked by SDS-PAGE under reducing conditions, and the degree of oligomerization is assessed by SDS-PAGE on a 3–10% gradient gel under nonreducing conditions. A typical yield of recombinant human MBP is 5 mg per liter of culture medium.

 The centrifugation of sheep erythrocyte suspension is performed at 350 × g for 5 min at 4˚C, and the cell density of an erythrocyte suspension can be determined by measuring the absorbance at 541 nm after the erythrocyte suspension has been totally lysed with water.

 The sheep erythrocytes are washed with saline three times and are then resuspended in saline at 1:20. 1 mL of a 200 μg/mL mannan solution is mixed with 1 mL of a 0.5 mg/mL CrCl₃ solution. The mixture is added to 2 mL of a 1 × 10⁹ cells/mL erythrocyte suspension, followed by incubation for 5 min at room temperature with occasional mixing. The reaction is stopped by adding 3 mL of ice-cold GVB. The resulting erythrocytes coated with mannan (mannan-erythrocytes, ME) are washed with GVB three times and then resuspended at a density of 1 × 10⁹ cells/mL.

 0.1 mL of the ME suspension prepared in the previous step and 0.4 mL of recombinant human MBP diluted with GVB are mixed and incubated with gentle shaking for sensitizing the ME with MBP.

 The ME suspension is then washed with ice-cold GVB and resuspended at a density of 1 × 10⁹ cells/mL. Two volumes of CH₅₀ of MBP-depleted guinea pig complement, 0.1 mL of the ME suspension sensitized with MBP, and GVB are mixed in a total volume of 1.5 mL on ice and are then incubated for 1 h at 37˚C.

 After the reaction mixture has been centrifuged, the absorbance at 541 nm of the supernatant is measured. Maximal lysis is obtained by the incubation of 0.1 mL of the ME suspension with 0.14 mL of water. The degree of specific lysis is expressed as a percentage of the maximal lysis.