[The construction and immune response of fusion DNA vaccine CRT180/HPV6E7].
OBJECTIVE
To investigate the immune response, in particular, the anti-virus cellular immune response induced by the constructed fusion DNA vaccine CRT (Calreticulin) 180/HPV6E7 in vivo and vitro.


METHODS
The HPV6E7 open reading frame was amplified by polymerase chain reaction from pUC/HPV6 plasmid. The CRT180 gene was cloned by reverse transcription from human muscle tissues. The PCR products of CRT180 and HPV6E7 were subcloned into pcDNA3. 1-GFP eukaryotic vector. The recombinant plasmids CRT180/ HPV6E7 was authenticated by restrict enzyme digestion and confirmed by DNA sequencing. The DNA plasmid HPV6E7 with report gene GFP was transfected to murine B16 cells by lipofectamine kit to establish the target cells. The HPV6E7-expressing cell line was selected by G418 and identified by RT-PCR. The C57BL/6 mice were vaccinated via intramuscular injection in the right legs with 100 microg plasmid encoding CRT180-HPV6E7, CRT180 or HPV6E7, empty plasmid without insert, and PBS group respectively. The changes of the T lymphocyte subsets in the peripheral blood of the mice were evaluated by flow cytometric analysis. The lymphocytes from the spleen and lymph nodes were harvested and co-cultured with HPV6E7-expressing cell lines. The CTLs kill activity and the cytokines IL-2, IFN-gamma secretion levels of the lymphocytes were assessed by LDH and ELISA respectively.


RESULTS
The constructed recombinant plasmids pcDNA3. 1-CRT180/HPV6E7, pcDNA3. 1-CRT180 and pcDNA3. 1-HPV6E7 were authenticated by restrict enzyme digestion and confirmed by DNA sequencing. Green fluorescence located in the cellular nucleus and plasma could be detected by fluorescent microscope after transfection with plasmids. The results of RT-PCR from the selected positive cell line showed the expected fragments of HPV6E7 mRNA. After immunization, the percentage of CD8+ or TCRgamma delta tau cells in the peripheral blood, the CTLs kill activity of the spleen cells and lymphocytes against HPV6E7-expressing cells, and the secretion levels of IL-2, IFN-gamma in CRT180/HPV6E7 vaccinated group increased significantly compared with the control groups (P < 0.05).


CONCLUSIONS
Vaccination with fusion DNA vaccine CRT180/HPV6E7 could elicit a more efficient HPV6E7-specific immune response than with HPV6E7 plasmid, indicating the potential of CRT180/HPV6E7 vaccine to enhance the antigen presentation. The recombinant CRT180/HPV6E7 might help the elimination of virus in animal models and accordingly be used as a vaccine candidate in the therapy of CA.
Zhao
Ke-jia
KJ
Department of Dermatology and Venereology, Sir Run Run Shaw Hospital, Zhejiang University Medical School, Hangzhou 310016, China.
Cheng
Hao
H
Xu
Yan
Y
Zhu
Ke-jian
KJ
Chen
Ming-li
ML
Zhang
Xing
X
Song
Tao
T
Wang
Qi
Q
Chen
Da-fang
DF
D000818:Animals; D017951:Antigen Presentation; D037282:Calreticulin / Q000235:genetics*; D045744:Cell Line, Tumor; D052163:Human papillomavirus 6 / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016176:T-Lymphocyte Subsets / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000235:genetics* / Q000276:immunology*
D037282:Calreticulin; D007376:Interleukin-2; D009856:Oncogene Proteins, Viral; D019444:Vaccines, DNA; D014614:Vaccines, Synthetic; C062495:oncogene protein E7, Human papillomavirus type 6; D007371:Interferon-gamma