Anti-tumor Effect Of Recombinant Invasive Lactobacillus Plantarum With Trichinella Spiralis And Lewis Lung Cancer Associated Antigen Gene SHSPs

Lung cancer is currently the cancer with the highest mortality rate.The morbidity and mortality rate continues to rise,and the age of onset is gradually younger.At present,the treatment methods for lung cancer mainly include surgery,radiotherapy,chemotherapy and biological immunotherapy.Biological immunotherapy mainly includes antibody therapy,cell therapy and tumor vaccine.In the process of tumor vaccine research,it was found that the live bacterial carrier vaccine has an obvious anti-tumor effect.Among them,Lactobacillus are probiotics,which have immunomodulatory ability and certain anti-tumor activity.Studies have shown that Lactobacillus can be used as vaccine carriers for the prevention and treatment of cervical cancer.In recent years,it has been discovered that certain parasitic infections can be anti-tumor,and Trichinella spiralis(T.spiralis)had obvious curative effect on many kinds of tumors.The laboratory found that T.spiralis and Lewis lung cancer-associated antigen sHSPs antiserum had a good anti-lung cancer effect.Therefore,this experiment constructs the recombinant invasive Lactobacillus plantarum with associated antigen gene sHSPs to study its effect on Lewis lung cancer.In order to provide novel ideas for tumor prevention and treatment,and to provide experimental data for the development and application of tumor vaccines.Construction of recombinant invasive Lactobacillus plantarum with T.spiralis and Lewis lung cancer associated antigen gene sHSPs.The sHSPs were amplified with specific primers and connected to the eukaryotic plasmid pValac.The recombinant plasmid was transfected into 293T cells to verify the expression.The validated recombinant plasmid was electroporated into the recombinant invasive Lactobacillus plantarum NC8/FnBPA.The positive clones were selected and named NC8-sHSPs.The growth characteristics,adhesion,invasion,colonization ability and safety of recombinant Lactobacillus plantarum NC8-sHSPs were tested.The results showed that the associated antigen gene sHSPs was successfully amplified.The constructed recombinant plasmid pValac-sHSPs can be expressed in eukaryotic cells.Analysis of growth characteristics showed that the growth characteristics of recombinant Lactobacillus plantarum NC8-sHSPs did not change significantly,and they could pass through simulated gastric juice in the form of viable bacteria,and could reproduce in simulated intestinal juice.The colonization test showed that NC8-sHSPs can adhere to and invade mouse mode-k cells in vitro,and can also be colonized in the small intestine.Safety testing showed that NC8-sHSPs had no significant effect on the proliferation of mode-k cells,and did not cause excessive release of pro-inflammatory cytokines such as IL-1β,IL-6,IL-12,IFN-y,and TNF-αin vivo or in vitro.And it did not cause significant changes in the weight of mice and significant damage to tissues and organs.The preventive effect of recombinant invasive Lactobacillus plantarum NC8-sHSPs on Lewis lung cancer.Firstly,1×109 cfu NC8-sHSPs was orally immunized,and 2×106 Lewis lung cancer cells were injected subcutaneously 7 days after the second immunization.After 14 days of tumor-bearing,the mice were sacrificed,the tumor was stripped,and the tumor inhibition rate was calculated.Immunological indicators were detected by ELISA and flow cytometry.CCK-8 and LDH assays were used to study the effect of recombinant sHSPs on the proliferation of splenic lymphocytes in vitro and the ability of inducing CTL to kill Lewis lung cancer cells.Finally,the histopathological damage was observed.The results show that recombinant Lactobacillus plantarum NC8-sHSPs can significantly inhibit the growth of Lewis lung cancer,with an average tumor volume and weight inhibition rate of 62.36%and 68.37%,respectively.Immunological testing found that NC8-sHSPs can induce sHSPs-specific IgG,increase serum levels of TNF-α,IFN-γ,total sIgA and intestinal lavage fluid levels of total sIgA.And Increasing the number of CD8+T lymphocytes,indicating that NC8-sHSPs can activate the host’s humoral immunity,cellular immunity and mucosal immunity to inhibit the growth of Lewis lung cancer.Lymphocyte proliferation test showed that recombinant sHSPs can promote the proliferation of mouse splenic lymphocytes,and combined with IL-2 can induce the production of CTLs that directly kill Lewis lung cancer cells.Immunohistochemistry showed that the expression of PCNA in the tumor was decreased,and histopathological observation showed no obvious damage.The therapeutic effect recombinant invasive Lactobacillus plantarum NC8-sHSPs on Lewis lung cancer.Firstly,2×106 Lewis lung cancer cells were injected subcutaneously to establish a tumor-bearing mouse model,and then treated with 1×109 cfu NC8-sHSPs for 15 consecutive days.The tumor was dissected after the treatment and calculated the tumor inhibition rate.ELISA was used to detect immunological indicators.Finally,HE staining was performed on tissues and organs to observe histopathological damage.The results show that recombinant Lactobacillus plantarum NC8-sHSPs treatment can also inhibit the growth of Lewis lung cancer and prolong the survival time of tumor-bearing mice.The average tumor volume and weight inhibition rates are 40.76%and 44.22%,respectively.Immunological test results show that NC8-sHSPs can induce sHSPs-specific IgG,increase serum levels of TNF-α,IFN-γ,total sIgA and intestinal lavage fluid levels of total sIgA,suggesting that NC8-sHSPs treatment can induce humoral immunity,enhance mucosal immunity and cellular immunity to kill Lewis lung cancer.HE staining of tissues and organs showed that there was no obvious pathological damage after treatment with recombinant Lactobacillus NC8-sHSPs.