Expression of bovine (Bos indicus) interleukin-18 in Escherichia coli and its biological activity
Authors: Kotla Siva Reddy
Golla Ramalinga Reddy
Veluvarthy V S Suryanarayana
Dowlathabad Muralidhar Rao
Hosur Joyappa Dechamma
Journal: Microbiology and Immunology (2010)
Discipline: MICROBIOLOGY
Keywords: IL-18; immune functions; IFN-γ production; Th1 immune responses; Bos indicus
Affiliations: Indian Veterinary Research Institute, Bangalore-560024, Karnataka, India.

Abstract:  IL-18 modulates immune functions by inducing IFN-γ production and promoting Th1 immune responses. In the present study, we amplified and cloned the sequence (582 bp) encoding full-length bovine IL-18 from PBMC stimulated with PHA. The nucleotide and the deduced amino acid sequence of Bos indicus IL-18 showed an identity of 86-98% compared with IL-18 sequences of other ruminants. The insert was subcloned into a pET 32a vector and expressed in Escherichia coli as a fusion protein and the matured protein was obtained by caspase I treatment. The specificity of these proteins was confirmed by western blotting. The biological activity of the purified protein was analyzed by its ability to induce IFN-γ production in PBMC measured by ELISA and qPCR. 

Retracted: No

PubPeer comments:
 Figure 2A: All bands in the marker lane look similar to each other. Shown with red arrows. ![file] This paper was found during a systematic screen of biomedical papers for image duplications, as published in Bik et al., mBio (2016), doi: 10.1128 mBio.00809-16, https: journals.asm.org doi 10.1128 mBio.00809-16. This issue was reported to the journal in October 2015, but it has not yet been addressed by the journal, more than six years later. , 
 --------- Forwarded message --------- From: Microbiology Immunology Asia mai@wiley.com Date: Thu, Nov 26, 2015 at 9:48 PM Subject: RE: Microbiology and Immunology:Regarding to your published article in Vol 54, Issue 9 To: Siva Reddy sivareddykotla@gmail.com Regarding to your published article u201cExpression of bovine (Bos indicus) interleukin-18 in Escherichia coli and its biological activityu201d published Microbiology and Immunology, 2010; 54(9): 564u2013567 Dear Dr Siva Reddy Kotla Thank you for your correspondence about this article. We have investigated the matter thoroughly and we are happy with your explanation that Figure 2A is not manipulated in appropriately. Again thank you for your cooperation. We look forward to receiving your new submission. Regards Microbiology and Immunology Editorial Office Wiley 155 Cremorne Street Richmond VIC 3121 Australia www.wiley.com T: 03 3830 1263 (Residents in Japan), +61 3 9274 3129 (Residents outside of Japan) F: FAX: +61 3 9274 3341 mai@wiley.com From: Siva Reddy [mailto:sivareddykotla@gmail.com] Sent: Friday, 16 October 2015 2:47 AM To: Microbiology Immunology Asia Subject: Re: Microbiology and Immunology:Regarding to your published article in Vol 54, Issue 9 Dear Editor Regarding Figure, 2A Marker lanes were look like very similar 1. We used 6 years old protein marker, which was got from Indian local company (Bangalore Genei cat no- 105978, lot number 5644326). Particular lot contained some problem all bands were similar look like they are diffused, means when you compared our figure 2b we can observe clear differences in each kilo Dalton mark (ex: see the difference between 17kDa and 45kDa) but in case where we used to particular these lot no difference between in each kilo Dalton bands. Further,we enquired with Bangalore Genei technical support team but they informed us 105978 product was discontinued. After, that we purchased the new marker from sigma, which was used in figure 2B. Bands were not similar when I checked my original figure in their differences were there but very little. Those all bands were diffused state. If you want us to send original figure we are happy to send. I hope you will satisfy above answer. Please let us know any question regarding this manuscript. We are happy to give answers. Thank you for your time and consideration. Youru2019s Sincerely Sivareddy Kotla On Thu, Oct 15, 2015 at 1:58 AM, Microbiology Immunology Asia mai@wiley.com wrote: Dear Dr. Kotla Siva Reddy Regarding to your published article u201cExpression of bovine (Bos indicus) interleukin-18 in Escherichia coli and its biological activityu201d, published in Microbiology and Immunology, 54, 564u2013567, It has been brought to our attention that one of the figures in the above manuscript may have been manipulated inappropriately(Please refer to the attached file). Figure 2 a: All bands in the marker lane look very similar We would be grateful for any explanation you can provide and look forward to hearing from you by October 23. Yours sincerely, Microbiology and Immunology Editorial Office Wiley 155 Cremorne Street Richmond VIC 3121 Australia www.wiley.com T: 03 3830 1263 (Residents in Japan), +61 3 9274 3129 (Residents outside of Japan) F: FAX: +61 3 9274 3341 mai@wiley.com , 
 Thank you for your reply, Dr. Kotla. I appreciate that you shared the correspondence with the journal editor from 2015. So you are saying that the similarities between the bands were caused because a particular lot of marker sample was problematic? This does not make any sense to me, but maybe you can explain in more detail. Usually, bands in a gel or blot are all slightly different from each other due to the presence of irregularities in the gel or during blotting, scratches on the developer rolls, blot wrinkles, etc. I cannot think of a technical reason why a particular batch of marker could behave like this on a gel or blot. You also offered to share the original photo with the journal editor, back in 2015. Would you still have that photo now to share here? , 
 We cloned bovine IL-18 into a PET32A expression vector; after induction, we purified the protein and estimated the protein concentration. 2.tRun the SDS-PAGE gel and transfer it to the PVDF membrane (Sample concentration -30 ug). 3.t5% Blocking (skim milk) buffer used and developed by using OPD Substrate and developed (we donu2019t have the image scanner). Dried up the membrane and scanned by the regular scanner. 4.tWe noticed did not the run marker properly. 5.tNext, we ordered markers from different vendors and changed the following using the ECL and image scanner (We used in other institution image scanners) 6.tAfter that, we repeated, with new markers, run them properly. But we used the first experiment figure. Thanks ![file] ![file] ![file] ![file] , 
 Thank you, Dr. Kutla for posting these photos. Looking at the higher resolution photo for Figure 2, my concerns have only gotten stronger. The marker bands look remarkably similar. This might be a concern because if there is doubt about the protein marker, it is unclear what the size of the band in Lane 2 might be. What was the Sigma marker s catalog number for the re-do of the experiment? ,