Cloning and Expression of N-CFTX-1 Antigen from Chironex fleckeri in Escherichia coli and Determination of Immunogenicity in Mice

Abstract

Background: Most jellyfish species are poisonous. Human victims of jellyfish sting each year are 120 million. Chironex fleckeri is a venomous box jellyfish that inflicts painful and potentially fatal stings to humans. The CfTX1 is one of the antigenic proteins of venom that is suggested to stimulate the immune system for treatment and vaccine. This study aimed to clone and express the CfTX-1 antigen in E. coli and then to determine the synthesis of related antibody in the mice.
 Methods: The study was performed in the Persian Gulf and Oman Sea Ecology Research Center, Bandar Abbas, Iran in autumn 2016. The synthetic CfTX-1 gene in PUC57 plasmid was purchased from Nedaye Fan Company. The 723 bp fragment of N-CfTX-1 was amplified by PCR, PUC57 plasmid containing CfTX-1 with BamHI SalI restriction enzyme sites were subcloned in pET28a [+] expression vector and transformed into E. coli BL21 (DE3). The CfTX-1 gene expression was induced by IPTG. Then antibody produced from the mice serum were isolated and confirmed by ELISA. After protein purification, resulted antigen was injected to mice in 4 repeats and then evaluated the rate of antibody in mice serum. Mice were challenged by the Carybdea alata.
 Results: The 726 bp of N-CfTX-1 were cloned in a vector of expression pET28a [+] and confirmed by PCR, sequencing and enzymatic analysis. Moreover, the recombinant protein was confirmed by SDS-PAGE and Western blotting. Then the antibody was isolated from mice serum and confirmed by ELISA test. The results showed that immunized mice tolerated 50x LD501 of jellyfish venom.
 Conclusion: The CfTX-1 recombinant protein was able to protect the BALB/c mice against jellyfish venom. The produced protein can be used as a candidate for vaccine against jellyfish venom.

Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for Stonustoxin Neutralization Studies

“…On the other hand, some recombinant venom components may present challenges, potentially due to their toxic nature, resulting in lower expression yields. For example, studies by JAFARI et al and Honari et al focused on the N-terminal segment of CfTX from Chironex eckeri (box jelly sh), yielding relatively low expression levels [45,46]. The lower expression yields in the case of recombinant toxins compared to other recombinant proteins can be attributed to their toxic nature.…”

Immunological detection of AcAMP antimicrobial peptide secreted by Aspergillus clavatus

“…In the first injection (day 0), 100 μl of “VAX-ORIENT IPA-70” adjuvant (Pars Vaccine Technology, Iran) was homogenized with 20 μg of recombinant peptide and mixed with PBS to a final volume of 200 μl. Similarly, in the next boosts (days 14, 28, 42), 20 μg of recombinant peptide homogenized with the mentioned adjuvant and were intraperitoneally injected to the animals ( Table 3 ) ( 16 , 17 ). Finally, we used prepared serum sample on day 49 due to its maximum antibody titer for the subsequent studies ( Fig.…”

“…To investigate the ability of the raised antibodies against the recombinant AcAMP and to detect the fungus-produced non denatured AcAMP, indirect ELISA was used ( 16 , 17 ). For this aim, a flat-bottom 96-well plate was coated with serially diluted crude protein starting at 10 μg in 100 μl coating buffer (15 mM Na 2 CO 3 , 36 mM NaHCO 3 , pH 9.8) and incubated at 4°C overnight.…”

“…Iran) was homogenized with 20 µg of recombinant peptide and mixed with PBS to a final volume of 200 µl. Similarly, in the next boosts (days 14, 28, 42), 20 µg of recombinant peptide homogenized with the mentioned adjuvant and were intraperitoneally injected to the animals (Table 3) (16,17). Finally, we used 2 4 mM Tris-HCl, 8 M urea, and pH 8) at 25°C for 1 hour and then centrifuged at 14000 rpm for 30 minutes.…”

“…8 M urea, pH 4.5). Finally, the column was washed with 1 ml of 250 mM imidazole (300 mM NaCl, 50 indirect ELISA was used (16,17) well. Finally, the reaction was stopped with 2.5 M H SO (100 µl/wells).…”