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ABSTRACT
In this study, purified GM-CSF and LMP2A mRNAs were amplified by PCR. Then, the GM-CSF and LMP2A sequences were connected by the polypeptide linker (Gly4Ser)3 using gene splicing by overlap extension. The constructed fusion gene GC2A was inserted into the adenovirus vector. Then the recombinant vector was introduced into HEK 293T cells by calcium phosphate transfection to package the adenovirus. The levels of antibodies against the GM-CSF and LMP2Afusion proteins were measured by ELISA, and the CTL activity of the mouse splenic lymphocytes was determined by lactate dehydrogenase (LDH) release assay. Immunotherapy of mouse tumor (EBV-positive epithelial tumor cell line (GT39)) tissues was performed, and their morphologies were assessed. Finally, the data of each group were analyzed using SPSS 11.5 statistical software. The recombinant adenovirus could replicate in HEK 293Tcells and induce humoral and cellular immune responses in the mice. The maximum dose resulted in an antibody titer of 18500 (184.5 ± 8.7 pg/ml). At an effector: target ratio of 40:1, maximum specific lysis was observed which was approximately three times that detected in the control immunized mice. The tumor inhibition rate was approximately 76% compared with the control groups, indicating the presence of significant differences among the groups. Tumor-infiltrating lymphocytes were detected by hematoxylin-eosin (HE) staining. The recombinant adenovirus induced humoral and cellular immune responses and inhibited tumor growth in mice. It provided a theoretical basis and candidate vaccine for further preclinical trials.
Abbreviations
| CTL | = |
cytotoxic lymphocyte |
| EBV | = |
Epstein-Barr virus |
| ELISA | = |
enzyme linked immunosorbent assay |
| GC2A | = |
GM-CSF and LMP2A fusion gene |
| GM-CSF | = |
granulocyte/macrophage colony-stimulating factor |
| GT39 | = |
EBV-positive epithelial tumor cell line |
| HEK | = |
human emborynic kidney |
| HE | = |
hematoxylin-eosin |
| LMP2A | = |
Epstein-Barr virus latent membrane protein |
| PCR | = |
Polymerase Chain Reaction |
| PBS | = |
phosphate-buffered saline |
| pAdTrack-CMV | = |
adenovirus shuttle vector |
| pAdEasy-1 | = |
adenoviral skeleton plasmid |
| RT-PCR | = |
reverse transcription polymerase chain reaction |
| SPSS | = |
Statistical Product and Service Solutions |
| SDS-PAGE | = |
sodium dodecyl sulfate polyacrylamide gel electropheresis |
Disclosure of potential conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgments
Hui Wang and Shuang Wang are thanked for their excellent technical assistance.
Funding
Research supported by NSFC cultivation project of Jining Medical University(2016), the Natural Science Foundation of Shandong Province (#ZR2012HM037,#ZR2014HL040), the Science and Technology Project of Colleges in Shangdong Province (#J12LK56,#J13LK54), and the National Natural Science Foundation of China (# 31500056, #30971081, #31271243, and # 81070961).
Authors' contributions
Ting Chen designed and conceived the study. Qing-jie Xue designed and performed the experiments. Chun-Mei Wang and Chun-qing Yang integrated the data and wrote the manuscript. All authors read and approved the final manuscript. All authors have been involved in revising the manuscript critically.
