Immuno-gene Therapy Of Superantigen SEB Gene For Mice Lewis Lung Cancer

Background and Objective: Lung cancer has become the No. 1 cancer killer throughout the world. In China, the incidence and mortality of lung cancer is still keeping rising. Although effect of treatment for lung cancer was improved to some extent during the past decades, because of the progress of in surgical technique, radio-chemotherapy, and application of TKIs treatment targeting against EGFR mutations, the overall 5-year survival rate is still less than 15%. So, it is of great importance to explore novel therapeutic approach for lung cancer. Immunotherapy for cancer has attracted attention for many years, and it has become one of the focus field in cancer research during the past years. One of the important strategies of immunotherapy for cancer is to transduce antigen gene into cancer cells to enhance tumor immunogenicity, so that strong immune response could be activated in and around the tumor cite. Super antigen is a family of small molecule antigen proteins produced by bacteria, viruses and fungi. Superantigen can activate fierce immune response at very small dose. Staphylococcal enterotoxins(SEs) is a big family of superantigens consists of SEA, SEB, SEC and other more than 10 subtypes. They are also the earliest, most deeply and extensively researched superantigens. As a kind of exotoxins secreted by bacterium, SEs are account for toxicosis caused by Staphylococci, such as fever, diarrhea and even toxic shock. However, some recent researches suggested that SEs could be used in cancer immunotherapy because of their strong antigenicity. In present research, SEB expression vector is to be constructed, and is used to treat Lewis lung cancer burdened mice. The research is aimed to explore feasibility and approach of immune gene therapy for lung cancer, using superantigen SEB gene.Methods: 1. Seeked for a template in Gen Bank, SEB gene is chemically synthesized with codon optimization. A pair of DNA primers are designed for SEB gene polymerase chain reaction(PCR) amplification as following: forward primer sequence: 5'- AAGTATCTAGAGATGCCACCATGTACAACAGACTCTTCGTCAGCC-3', reverse primer: 5'-GCCGAGGATCC TCACTTCTTCTTAGTTGTCAGGTATA-3'. We designed restriction sites for Xba I(TCTAGA) and Bam HI(GGATCC) at the terminal of 5' of the above primers. 2. The SEB gene sequence is cloned into expressive plasmid PCDH-CMV-GFP, so that SEB expression plasmid driven by CMV promoter. DNA sequencing and restriction enzyme digestion is performed to verify the recombinant SEB plasmid. 3. PCDH-SEB-GFP was transfected into Lewis lung cancer cells with Lipofectamine 2000, and SEB expression in tranfected Lewis lung cancer cells was detected with western blot. 4. Lewis lung cancer cells were subcutaneously inoculated into C57 mice on the right side of the axillary area(3 x 106 cells each mouse). When the tumor grow about 50 mm3 in size, all mice were divided into three groups(6 mice in each group) : the experimental group(intra-tumoral injection with PCDH-SEB-GFP), negative control group( intra-tumoral injection with empty vector PCDH-CMV-GFP) and blank control group( intra-tumoral injection of PBS). Plasmids were injected every 3 days and repeated for 3 times. 20 ug corresponding plasmid and 20?l lipofectamine was used in each injection in 100 ?l volume. The tumors were observed and measured with calliper every 2 days, so that the growth curve could be drawn. Two days after the last injection, blood of mice was collected with eye-ball sampling method. And then, all the mice were sacrificed with neck dislocation and tumor were wholly peeled off. Tumors were measured with calliper and weight was measured with electronic balance. The average tumor volume, weight of the 3 groups was compared. 5. Tumor tissue was HE stained for routine pathological study and immunohistochemistry(IHC) was performed to detect SEB expression in tumor cells. Enzyme-linked immunosorbent assay(ELISA) was used to detect concentration of inflammatory factors of Interferon-?( IFN-?) and tumor necrosis factor-?,(TNF-?). Results: 1. A 800 bp SEB gene was successfully synthesized, and its sequence was proved to be same as expectations by agarose gel electrophoresis and DNA sequencing. 2 The SEB expressive plasmid PCDH-SEB-GFP was successfully constructed. Western blot suggested that SEB could express in Lewis lung cancer cells, when PCDH-SEB-GFP was transfected with lipofectamine 2000 2 days later. 3. Tumor model of C57 mice was successfully established. PCDH-SEB-GFP therapeutic experiment revealed that average volume and weight in experiment group mice was significantly lower that negative and blank control groups. Pathological study revealed that massive necrosis of tumor tissue and infiltration of inflammatory cells around necrotic area could be observed in experiment group. IHC revealed that SEB expression in some tumor cells of experiment group mice was detected, while not in other 2 groups. ELISA study suggested that no significant difference of IFN-?and TNF-? concentration among the 3 groups was observed. Conclusions: 1. SEB gene transfected with liposome can be expressed in Lewis lung cancer cells. 2. Superantigen SEB gene can inhibit tumor growth in mice.