| Development of methane mitigation for ruminants will not only increase the utilization of feed energy, but also is meaningful for slowing global warming and protection of environment. The reverse vaccinology is used in this experiment to screen the gene wich encoded the member protein EhaF from the genes of Methanobrevibacter ruminantium Ml. We express the preotein EhaF in E. coil and study the effect on the rumen methane emission and rumen arahaea in goats after immunication of the product.We screen the mru 1407 from the genes of Methanobrevibacter ruminantium M1 which are suitable for cloning and expression. The mru 1407 was amplified from the DNA extracted from fresh rumen by PCR, then cloned into pET30a (+) to construct the expression plasmid pET30a (+)-mru 1407. The recombinant protein was expressed in E. coil Rossetta and purified by Ni-NTA, then was sequenced by mass spectrometer. Twelve goats were allocated into 2 groups of 6 each randomly. Each animal of vaccination group was injected intradermally with lml Elution Buffer containing 400ug purified protein emulsified with FCA, and boosters in FIA were given at days 35,49. The control group were vaccinated with 1ml Elution Buffer mixed with 1ml FCA/FIA at the same time. Sera, Saliva and rumen fluid were taken on day 63. After collecting all the samples, the animals were transferred into indirect open-circuit respiration chambers. An indirect ELISA was used to measure antibodies present in the sera and saliva. The test of blood count, blood chemistries were taken by animal hospital. The V4 DNA of archaea were amplified by PCR using V4 primer from DNA of rumen samples, then were subjected to sequencing by using Illumina plateform. QIIME was used to analyse the data to compare the species diversity and releative abundance of rumen archaea and methanogens between two groups.The results show that (1) The recombinant protein was expressed successfully in E. coil and purified by Ni-NTA. The result of mass spectrometry analysis showed a clear match between His-EhaF and predicted peptides. (2) The protein caused immune response after injection and the mean titers of serum and saliva were much higher than that in control group. (3) The injection of protein His-Eha didn’t effect the blood count, blood chemistries (P>0.05). (4) There is no marked difference of CH4 emission per dry matter intake between control and vaccination group (P>0.05). (5) The only phylum of archaea found in rumen of goats was Euryarchaeota, which were divied into six families: Methanobacteriaceae, Methan-omicrobiaceae, Methanosaetaceae, Methanosarcinaceae, Marine_Group_Ⅱ, vadinCA11_gut_ group. The Methanobacteriaceae and vadinCA11_gut_group family took the main percentage of rumen archaea, which is 57.40% and 37.8% respectively. The Methanobrevibacter is the main part of rumen archaea in genus level (57.40%). (6) There is no marked difference of species diversity and releative abundance of rumen archaea between control and vaccination group (P>0.05). (7) The injection of protein EhaF didn’t affect the relative abundance of methanogens:Methanobacterium, Methanobrevibacter, Methanomicro-bium, Methanosaeta, Methanimicrococcus (P>0.05).In conclusion, the member protein EhaF of Methanobrevibacter ruminantium Ml could be expressed correctly in E. coil. The immunization of recombinant protein His-EhaF could cause the high titers in serum and saliva and doesn’t affect the indexes of blood count, blood chemistries, rumen methane emission and species diversity and releative abundance of rumen archaea. In conclusion, the member protein EhaF of Methanobrevibacter ruminantium M1 isn’t a broad spectrum antigen for rumen methanogens. |
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