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| Establishment of a Novel Transgenic Insect Cell Line Displaying Cotton Bollworm (Helicoverpa armigera) Cadherin Protein on Membrane Surface |
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Abstract The purpose of this study was to establish a transgenic insect cell line containing Helicoverpa armigera cadherin (HaCAD) for subsequent study on the interactions between Bacillus thuringiensis (Bt) insecticidal crystal protein and its receptor cadherin. Based on the specific membrane-adhering characteristics of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) envelope protein GP64, PCR primers were designed based on AcMNPV gp64 gene sequence. A 172 bp DNA fragment of gp64 signal peptide (gp64sp) and a 135 bp fragment of the gp64 C-terminal transmembrane domain (gp64ctd) were amplified by PCR, respectively. Both gp64sp and gp64ctd fragments were ligated and an additional DNA sequence containing 6 restriction digestion sites was inserted. This 301 bp fragment was replaced into the multiple cloning sites of expression vector pIZ/V5-His. A surface display vector named pIZ/V5-gp64 that allowed the exogenous gene to be expressed on the cell membrane was successfully constructed. The amplified 3 882 bp of the cadherin repeat (CR) and the membrane proximal region (MPR) of HaCAD and recombinant plasmid pIZ/V5-gp64 were digested with restriction enzymes, respectively, and then ligated. A recombinant vector named pIZ/V5-gp64-HaCAD was constructed. This vector was transfected into cotton bollworm cell line Ha-E-1 via liposome-mediated transfection. The transfected cells were screened on the selective culture medium containing zeocin and the selected cells were further cloned. A transgenic cell line Ha-T-CAD containing the recombinant HaCAD gene was obtained. A majority of transgenic cells were circular in shape while a few cells exhibited short spindle shape. Compared with the original parent Ha-E-1 cells, no obvious changes in the both morphology and size of the transgenic cells Ha-T-CAD were seen. PCR verification results showed that 3 882 bp fragment of HaCAD gene was successfully integrated into these cells. The results of immunofluorescent visualization revealed that in the transgenic cell line Ha-T-CAD, the red fluorescent dye recognizing cadherin protein was mainly distributed in cells. Western blot analysis indicated that 140 kD of cadherin protein expressed and displayed on the membrane of Ha-T-CAD cells. This established transgenic cell line Ha-T-CAD could be used to study the interactions between Bt insecticidal crystal protein and its receptor cadherin and will provide an important research tool for studying the action mechanism of Bt insecticidal crystal protein and the mechanisms underlying the resistance of insects against Bt toxin.
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Received: 24 November 2015
Published: 01 April 2016
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