I have cloned all eight genes from H9N2 virus into PHW2000 plasmid successfully. All prepared midi plasmids were verified correct with Sanger sequencing except PHW-HA plasmid which gave me unexpected similarity against the reference HA sequence from my original virus? The similarity was about 77-80% with other HAs like H7 and NOT H9? Is there any solution for such problem? I used DH5 alpha in cloning all genes.
No clue. Since HA is shorter than PB1, PB2 and PA and you have correct sequence for them, I would say your cloning system is reliable (cloning error can not be 20-23%). Your original virus may be contaminated with an H7 HA fragment somehow.
My suggestion is that you can chemically synthesize the HA you want, again, as you already have 7 fragments ready.
I think it is unlikely to because of your E.coli strain. Either the original virus really has these sequences or something from the PCR (if you did so in your cloning strategy). Did you amplified the segments firstly using PCR or how did you do your cloning? If yes, which enzyme you used for PCR? Q5 or Phusion or Dream Taq?
Thanks, Ibrahim. Yes the problem was not from E-coli but from original virus (was not pure) so I did plaque purification and we solved the problem.
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