Acrylamide concentration affects the relative position of VP7 gene of serotype G2 strains as determined by polyacrylamide gel electrophoresis

Abstract

Background: During the course of development and characterization of various rotavirus reassortants, we found that the relative position of the gene encoding neutralization and protective antigen VP7 of certain rotavirus strains in a PAGE gel was influenced by the concentration of acrylamide.

Objectives: We investigated systematically various factors that affected the relative position of the rotavirus VP7 gene in a PAGE gel.

Study design: We analyzed dsRNAs of selected rotavirus strains bearing G1, G2, G3 or G9 specificity by PAGE at varying concentrations of acrylamide.

Results: We demonstrated that the relative position of the VP7 gene of three G2 strains varied depending upon the concentration of acrylamide in a PAGE gel, which occurred not only in a homologous G2 virus gene background but also in a heterologous G3 virus gene background; and the VP7 gene bearing G1, G3, G4 or G9 specificity did not display this phenomenon when the PAGE running conditions were varied.

Conclusions: The concentration of acrylamide in a PAGE gel was the major factor that influenced the relative position of the VP7 gene of G2 rotavirus strains (i.e., VP7 gene coding assignment by PAGE).

Figure 1

Electrophoretic migration patterns of each of four components of RotaShield™ and their parental rotavirus strains in 7.5% (A) and 15% (B) PAGE gels. Lane 1, D strain; lane 2, D × RRV; lane 3, RRV; lane 4, DS-1 strain; lane 5, DS-1 × RRV; lane 6, RRV; lane 7, RRV; lane 8, ST3 strain; lane 9, ST3 × RRV; lane 10, RRV. Arrowheads indicate the VP7 genes.

Figure 2

Electrophoretic migration patterns of Swedish G2 strain 1076, 1076 × RRV reassortant and D × RRV in 10% (A) and 15% (B) PAGE gels. Lane 1, 1076 × RRV; lane 2, D × RRV; lane 3, 1076 strain; lane 4, 1076 × RRV. Arrowheads indicate the VP7 genes.

Figure 3

Electrophoretic migration patterns of Japanese G2 strain S2, S2 × RRV and D × RRV in 10% (A) and 18% (B) PAGE gels. Lane 1, S2 × RRV; lane 2, D × RRV; lane 3, S2 strain. Arrowheads indicate the VP7 genes.

Figure 4

Electrophoretic migration patterns of DS-1 strain, DS-1 × RRV and RRV in a 5% regular (18 × 16 × 0.075cm) (A) or 5% long (18 × 32 × 0.075cm) (B) PAGE gel. Lane, 1, DS-1 strain; lane 2, DS-1 × RRV; lane 3, RRV. Arrowheads indicate the VP7 genes.

Figure 5

Secondary structure of VP7 gene mRNA of strain D (G1); strains DS-1 (G2) and S2 (G2); strain RRV MMU18006 (G3); and strain 116E (G9) as predicted by GeneQuest program.