Preparation of Bacterial strains and its culture
Mycobacterium bovis strains lyophilized and provided from Pasteur Institute of Iran, Department of BCG vaccine preparation with number 11732:P and then cultured in three stages of seeding SP, S1, S2, cultured to Sauton’s minimal medium (Sauton’s) and incubating for three weeks at 37 ◦C.
Preparation of the first passage (SP): includes the removal of a vial of primary or secondary seeds of bacillus from the lyophilized strain bank. After measuring the vacuum of the seed vial, using a spark device, and ensuring the existence of a vacuum, the contents of the vial or Sauton are suspended in a thick suture and transferred in equal proportions on the surface of the potato inside the tubes for 14 to 22 The day was incubated.
Preparation of the second passage (S1): This stage was performed 14 to 22 days after inoculation of the first stage (SP). One of the tubes containing the first-stage growth bacterium (SP) that was approved was removed along with the flasks containing the concentrated Sauton culture medium and placed in a flask with a platinum ring on the surface of the Sauton inside the flask. Incubation was performed for 6 to 8 days.
Preparation of the third passage (S2): Selection of the number of Fornbach flasks containing grown bacteria of the second stage (S1) which was approved along with the desired number of flasks containing concentrated sap was selected. With a platinum ring, some of the bacteria grown on the surface of the bun were taken and placed on the surface of the bun in a flask. Incubation was performed for 6 to 8 days.
Stages of preparation of BCG bacillus cake involve the separation of BCG bacillus from the Sauton culture medium (filtration operation) using Birko and preparation of BCG cake: The contents of the flasks are gradually and continuously poured into the funnel and washed with phosphate buffer. After the solution was completely removed from the Birko output, the beaker opening (pre-weighed) was placed under the Birko outlet, and the BCG cake was transferred into the beaker, and the weighing step was performed. Weighing and measuring the moisture content of the BCG bacillus cake was calculated, and then the cake was transferred to 2-8 ° C. The lipids are then extracted in a 1: 2 ratio of chloroform-methanol.
For LAM / LM purification, the cells were resuspended in deionized water and centrifuged twice at 6760 rpm for 20 min at 25 ° C. Biomass was again separated from the supernatant for 18 hours at 18,000 rpm in the centrifuge at 25 ° C. Glucans, proteins, DNA and RNAs contaminated with enzymatic degradation were removed using α-amylase, trypsin, DNase 1 and RNase treatments followed by dialysis. Next, an equal volume of 90% phenol was added to the liquid containing lipoglycan, and the mixture was incubated by shaking at 68 ° C for 1 hour. The resultant solution was exposed to dialysis saline for 24 hours, during which time the physiological serum was changed twice during this period.
Data design and modeling were performed at three levels with three factors: chloroform/methanol, centrifuge speed, and pH using mini-tab software in nine shifts. To design the variable amount of chloroform/methanol in three levels in equal proportion, 1 to 2 ratio and 2 to 1 ratio were designed. The pH variable was designed in three levels of 6.5, 7, and 7.5 and speeds of 6000,6500,7000 rpm.
Optimization of significant variables was performed by the response level method in the form of a central composite design by Minitab software version 17.
For purification of mannan using Sephadex G75 chromatographic column, the mannan content solution was passed through Sephadex G75 2.5 x 14 column under laboratory temperature conditions. The exit rate was 1 ml/min, and 18 microtubes were collected.
The toxicity of the extracted mannan is assessed under two conditions.
Evaluation of in vitro toxicity by MTT test
The toxicity of the extracted mannan on MDA-MB-231 cells and normal cell lines was evaluated by MTT assay.
Cells with a density of 104 were cultured on plate 96 cells in an incubator at 37 ° C in the presence of carbon dioxide. After 48 hours, the cells were dissolved in different amounts of mannoprotein in PBS (Phosphate buffer Solution). After specified periods (24 hours and 48 hours), the culture medium was replaced with a new culture medium containing MTT solution. DMSO (Dimethyl Sulfoxide) was then added to each well and the absorption of foramazone at 490 nm was measured using an ELISA device. After the MTT test, the viability of MDA-MB-231 cells on the y-axis and different concentrations of mannoprotein on the x-axis are plotted to assess the toxicity. Then the IC50 value was calculated by the point-to-point method using Excel software (Chang et al. 2007).
Concentrations of 250, 125, 62, 31, 15, and 500 μg / ml were performed for the two cell lines with the extracted mannan and the test results were evaluated comparatively. Dilution is performed in a ratio of one to two dilutions so that no more cytotoxic effects are observed. Dilution liquid samples start from a quarter dilution. The IC50 is calculated if the sample is cytotoxic. The rate of cytotoxicity is obtained by reducing the percentage of living cells from 100%.
Abnormal Toxicity Test in Rodents
In this test, more than 5 healthy male SW1 mice weighing between 17 and 24 g were selected. More than two healthy male guinea pigs weighing between 250 and 400 g were chosen. The clinical condition of the animals was monitored in terms of health by a veterinarian. At the end of the adaptation stage (24 to 48 hours), among the isolated mice, five healthy same-sex mice were put in a type II cage containing a sterile bed. Furthermore, two same-sex guinea pigs were placed in a type IV cage (in the general method in BP pharmacopeia, the test sample was injected only on mice). The cages of the picked mice and piglets were moved to the test room. The animal was given one to two hours to decrease the caused transportation stress.
Based on the method of antiserums and vaccines, the test sample should be one human dose, provided that it is not more than 1 ml, for each mouse and one human dose, provided that more than not more than 5 ml, for guinea pigs, was injected intraperitoneally. In the general pharmacopeia method, the animals were monitored for 24 hours; in the antiserum and vaccine method, the animals were monitored for seven days. During the seven days after injection, clinical examination was performed at least once a day, and all symptoms or changes in animal behavior were recorded. After seven days of injections, each animal was weighed. The difference between weight at injection time and final weight (after seven days) was calculated.
Immunogenicity test
To assess the immunogenicity, antigens were injected intraperitoneally on 133 mice. The first group was injected with 0.5 micrograms of mannan. The second group was injected with 0.5 micrograms of mannan and BCG vaccine. The third group was injected with the BCG vaccine. The fourth group was assigned as the control group. Blood sampling was performed in the fourth week after injection. Blood was centrifuged at 3500 rpm. The sera were separated and stored in the freezer. After blood sampling and serum preparation, the immunogenicity of antibodies including IgG, IgM, and interleukin 12 was evaluated to compare the BCG vaccine and BCG vaccine with mannan and mannan alone.
Findings
Toxicity test results
To evaluate in vivo toxicity, the sample is approved in the general pharmacopeia method if all mice survive after 24 hours. In this study, mice were still alive after 24 hours.
According to Table 1, no significant decrease in weight and mortality was observed in the study animals.
[Table 1 near hear]
MTT: If there is no significant difference between the control and treatment groups regarding cell growth, it can be concluded that the device has no cytotoxicity. Nevertheless, if there is a notable reduction in cell life by the MTT method in the treated cells, it can be concluded that the tested device has cytotoxicity. The results in Figs. 1 to 4, no significant difference was observed between the control group and the treatment group in terms of cell growth. It can be concluded that the extracted MTT mannan is free of cytotoxicity.
[Figs. 1-4 near hear]
Immunogenicity results
The results in Tables 2 to 5 indicate that the level of antibody and interleukin 12 in the combination of BCG vaccine and mannan show good immunogenicity.
[Table 2-5 near hear]