Production of Recombinant protein Fim-C Salmonella typhi
The characterization of production and purification recombinant protein Fim-C Salmonella typhi show the presence of a single band on Lane E on Fig. 1 with ± 31 kDa molecular weight. These results concluded that the process of overexpression and purification of the Fim-C Inclusion Bodies S. typhi protein has been successfully performed [14, 15, 27].
Activity and Specificity of Recombinant protein Fim-C Salmonella typhi
Developing band with brown color was the results of oxidation reaction between peroxide's substances (H2O2), with catalyzed by Horse Radish Peroxidase (HRP) enzyme. The Horse Radish Peroxidase (HRP) enzyme changed DAB (3, 3'-Diaminobenzidine or 3,3',4,4'-Biphenyltetramine) substrate to Quinone Iminium substance, which is radical properties. Afterward, the Quinone Iminium substance induced the occurrence of polymerization reaction to form polymer substance with bigger molecular size and produce precipitate with brown color [9, 14, 18, 21]. The condition of antigen-antibody interaction on Fig. 2, was respectively as follows (1) The concentration of antigen is 3 µg, (2) the 100-time dilution of anti-recombinant protein of Fim-C S. typhi antibody, and (3) the 5000-time dilution of secondary antibody anti-mice labeled with the Horse Radish Peroxidase (HRP) enzyme. This result proves that Fim-C-S. typhi antigen has successfully identified its specific Anti Fim-C-S. typhi antibody.
Acute Toxicity Test of Recombinant protein Fim-C S. typhi
Physiological Health Analysis of ddY Mice
The clinical observation until day 14 of post-acute toxicity test showed that giving recombinant protein Fim-C-S. typhi with 25 µg concentration did not show any physiological disorder to central nervous system of ddY mice. It is such as the influence of analgesia and decrease of cognitive sharpness (sedation), trembling or seizure (tremors and convulsions), and motor activity (the mice remain active hanging on the roof of the cage and often sniff around the cage/have a high curiosity).
The male and female ddY mice immunized with a dose of 125 µg protein Fim-C-S. typhi experience symptoms such as sedation, convulsions and tremors, eyelid opening and abnormal breathing rates as well as tail standing in some of the mice. Three hours’ post-injection of antigen Fim-C-S. typhi with 125 µg concentration, three males and females in Sample Group 2 (KS-2) experienced mortality and two mice of each group still can survive until 14 days of the intensive observation period.
The male and female ddY mice immunized with a dose of 250 µg protein Fim-C-S. typhi experience symptoms such as sedation, convulsions, tremor, eyelid opening and abnormal breathing rate as well as tail standing in some of the mice. One-hour post-injection of Fim-C-S. typhi 250 µg concentration, three males and females ddY mice from Sample Group experienced mortality.
Analysis of Weight Change in ddY Mice
The evaluation results of body weight measurement of ddY mice in the control group and sample groups in general experienced weight gain. This indicates that either mice feed pellet or Fim-C-S. typhi protein test had the same effect to weight gain of mice. We can conclude that the giving Fim-C-S. typhi didn’t give significant change of appetite the animal test.
The evaluation results indicate that the weight is gain post immunization. The blue line shows the weight before treatment, and the red line indicated weight after treatment.
Analysis of Changes in Body Temperature of ddY Mice
The mice on sample groups (KS) experienced increased body temperature on the first day-after injection of Fim-C-S. typhi recombinant protein. It is caused by the body's response to the entry of foreign substance (Fim-C protein) into the mice body. The Fim-C protein will act as an antigen affecting the immune system and stimulating the leukocytes to release interleukins that will directly the set point thermoregulators in the hypothalamus [10, 19]. The body temperature of the mice ranges from 34.9–35.2 oC with an average body temperature change (ΔT) of 0.1-0.3oC. Increase in body temperature of mice is still considered normal if it is not more than 1oC.
The result of body temperature changes evaluation of ddY mice was proved through statistical data processing by one-way ANOVA. It showed the significance level P > 0.01, where the null hypothesis (Ho) is accepted, and Alternative Hypothesis (Ha) are rejected. The results showed that there was no significant difference in temperature changes (ΔT) between sample and control groups of mice (KS-1, KS-2, and KN). Sequentially P-value of the male and female mice in sample group 1 (KS-1) was 0.836 and 0.917, while in sample group 2 (KS-2) was 0.908 and 0.779 [23]. The results of statistical data processing with one-way ANOVA conclude that Fim-C-S. typhi recombinant protein with concentration of 25 micrograms (∝g) and 125 micrograms (∝g) had no significant effect on temperature changes of the both ddY mice.
Analysis of Mass Organ Changes in ddY Mice
Based on the organ mass data in Table 2, the statistical data was processed using one-way ANOVA of kidney, heart and spleen have results below:
a. Kidney. The statistical data processing showed significance level P > 0.01, which means no significant effect of Fim-C- S. typhi protein on the changes in kidney mass of ddY mice.
b. Heart. The statistical data processing showed the significance level of P > 0.01, which means there is no significant effect of Fim-C-S. typhi protein on changes in liver mass of ddY mice.
c. Spleen. The results showed that spleen in the mice sample group that has been immunized with the Fim-C-S. typhi protein works harder to produce antibodies in response to the presence of Fim-C-S. typhi recombinant proteins in the body. The results of statistical data processing of spleen organ in male and female ddY mice with a one-way ANOVA show significance level for sample group 2 and 3 (KS-2 and KS-3) of P < 0.01, while sample group 1 (KS-1) shows the significance level of P > 0.01. Therefore, sample group 2 and 3 are applied null hypothesis (Ho) refused and alternative hypothesis (Ha) accepted, which means there is a significant effect of giving Fim-C-S. typhi recombinant protein with 125 µg and 250 µg concentration to change spleen organ masses in both ddY mice. Meanwhile, sample group (KS-1) that was giving Fim-C-S. typhi recombinant protein with 25 µg concentration did not give significant effect to change spleen organ mass of male and female ddY mice.
Based on Table 3, mortality case was found in groups of mice immunized with Fim-C-S. typhi protein concentrations of 125 and 250 µg /mL; mortality rates in each group are 60% and 100%. Based on the acute toxicity test of recombinant protein of Fim-C S. typhi in Table 3, a curve of LD50 dosage calculation depicting the relationship between doses of Fim-C S. typhi protein with the mortality rate of ddY mice is shown in Fig. 8.The linearity that describes the relationship between dosages of Fim-C-S. typhi protein with mortality of ddY mice yielded the equation of the line y = 0, 4258x − 2.5806 with regression value equal to 0, 9758. From the equation of the lines, it can be determined LD50 dose by converting the value of x so that obtained LD50 dose (dose that can cause mortality) capable of 123.486 µg/mL. The results of the evaluation of acute toxicity test also showed that the safe dose range of Fim-C S. typhi recombinant protein for male and female ddY mice is at range 25–120 µg/mL.