Abstract: Study on the impact of intestinal microbial ecology of host animals by mucosal immunity with attenuated invasived bacteria，based on the 16S rRNA gene sequences of three cellulolytic bacteria including Ruminococcus albus,Ruminococcus flavefaciens and Fibrobacter succinogene,the primers were designed.Ruminal microbial total DNA was isolated from rumen content，and amplified by PCR.The PCR products was linked with pMD-18T Vector to construct recombinant plasmid and transfected into Escherichia coli.Target plasmids were selected by blue-white selection and their specificity was identified by direct PCR and DNA sequencing.The serial gradient concentrations of plasmid DNA were used to Real-time PCR.Results showed that neucleotide homology of 16S rRNA of three cellulolytic bacteria were higher than 99%.There were obvious differences in amplification curves of Real time PCR by the gradient concentrations of the recombinant plasmid，the correlation coefficient of the three standard curves were close to 1.The melting curves of Real-time PCR displayed a single peak.It was indicated that the standard plasmids and curves were constructed successfully and can be applied to the future research of mucosal immunity with attenuated Salmonella bacteria.

Key words: rumen cellulytic bacteria; Real-time PCR; standard curve