Study On Specific Labeling Of Enterotoxigenic E.coli By Red Fluorescent Protein Gene

It is necessary to establish specific detection method based on a specific pathogen,which can be used as a general indicator for the establishment of a model of intestinal challenge in vivo.However,the model labeled with green fluorescent protein(GFP)has some problems,the strain is not genetically stable under relaxing the selection pressures,and detection specificity of strains is not strong.Because the green fluorescence is greatly interfered by the autofluorescence in the animal,the result is that the fluorescence measurement accuracy is not high.In this study,ETEC was used as a general pathogen,and red fluorescent protein labeling method was used to try to apply different molecular labeling patterns.It is proposed to construct a fluorescent labeling strain with stable fluorescence expression and specificity,in order to further establish a pathogenic bacteria challenge model.The model lays the foundation for strain and methodology.The research content and related results are as follows:(1)The red fluorescent proteins mKate and mCherry were selected as reporter genes,and were linked to expression vectors pET-28a,pET-9a and pQE-30,respectively,to construct six recombinant plasmids.The recombinant plasmids pQE30-mKate and pQE30-mCherry,which can be judged by the obvious change of cell color on the next day of transformation,were successfully constructed without adding inducers.The two plasmids were transferred to different ETEC(E.coli K88ab,E.coli K88ac,E.coli K99 and E.coli 987P),and the recombinant strain E.coli K99-mCherry was obtained by observing the color change of the transformed strain with naked eyes,detecting the fluorescence intensity and performing enzyme digestion verification screening.The results show that the successfully constructed recombinant plasmid pQE30-mCherry can be efficiently expressed in E.coli K99without induction,and the overexpressed mCherry leaks out of the cell,which is convenient for intuitive selection of transformants.PCR confirmed that the expression of pathogenic factors has not changed and the growth characteristics have not been affected in the strain labeled with mCherry gene.Obvious red fluorescence can be observed under a fluorescence microscope.By establishing the calibration curves of bacterial counts and fluorescence intensity from E.coli K99-mCherry(R~2=0.0.9998),the detection limit of cell concentration was 10~3 CFU/mL.(2)In order to further optimize the expression of foreign protein mCherry,it is proposed to increase the fluorescence expression of the strain by increasing the copy number of its gene.Two copies of recombinant plasmid of mCherry gene were constructed by means of multi-copy expression cassette and multi-copy tandem of target gene,and the recombinant plasmid was transformed into E.coli K99 for heterologous expression.Then the fluorescence of bacteria was measured to determine the optimal copy number of mCherry gene expression.The results showed that the fluorescence intensity of the two copies of recombinant E.coli K99-2×mcherry and E.coli K99-×2mchery were lower than that of E.coli K99-mCherry,so the expression effect of single copy mchery gene in E.coli K99was the best.(3)This experiment explores the method of labeling ETEC with mCherry gene on the surface,which is to integrate Lpp and OmpA as anchoring proteins by over-lap PCR to construct recombinant plasmid pQE30-Lpp'OmpA-mCherry,and then transform into host E.coli K99.The labeled strains were evaluated by microscopy,molecular identification,growth characteristics analysis and stability of fluorescent protein expression.The results showed that the pathogenic genes of the recombinant strain E.coli K99-Lpp'OmpA-mCherry were not lost,and the growth characteristics were not changed.The growth of the cells was synchronous with its fluorescence signal,and the genetic stability of fluorescence expression was higher.Red positive clones were observed on the transformation plates without induction,and significant red fluorescence was observed under a fluorescence microscope.This study demonstrates the constitutive expression of the foreign protein mCherry in ETEC.By establishing the calibration curves of bacterial counts and fluorescence intensity from E.coli K99-Lpp'OmpA-mCherry(R~2=0.9997),the detection limit of cell concentration was 10~4 CFU/mL.(4)Based on CRISPR/Cas9 technology,E.coli K99 is used as pathogenic bacteria to carry out mCherry gene site-specific insertion to realize E.coli K99specific labeling.Based on the sequence of E.coli K12 gene,the pseudogene yheO site into which foreign gene can be inserted was selected.According to the selected pseudogene site sequence,sgRNA was designed and pTargetF-sgRNA vector was successfully constructed.The left and right homologous arms of yheO were amplified using pseudogene yheO as template.T5 promoter,target gene mCherry,left and right homologous arms and linearized fragment J23119(Spe I)-sgRNA-gRNA scaffold were linked by overlapping extension PCR through the design of primers.Finally,the gene editing vector Donor containing exogenous gene mCherry was constructed by linking reaction with Kpn I and Sph I double enzyme cutting linearized vector pUC57.The successfully sequenced Donor vector was transformed into E.coli K99-Cas9 competent state,and the positive clones which were successfully verified were added to the inducer IPTG,cultured at 30?overnight,and cultured at 42?for16 h to remove the gene editing vector.The results showed that PCR confirmed that the fixed-point knock-in of mCherry did not affect the growth characteristics and pathogenic factor expression of E.coli K99,the growth of thallus was synchronous with the expression of fluorescence,and the fluorescence expression was very stable.Under the induction of inducers,single colony showed visible red,and obvious red fluorescence could be observed under fluorescence microscope.Alpha lactose can replace the common inducer IPTG,and when the concentration is 5 g/L,the fluorescence expression of the bacteria can reach a higher value.By establishing the calibration curves of bacterial counts and fluorescence intensity from gene editing strain(R~2=0.9999),the detection limit of cell concentration was 10~4 CFU/mL.(5)By comprehensively comparing the expression stability of the fluorescent gene mCherry of the three recombinant strains constructed previously,an optimal marker strain was screened for specific detection of fluorescence in rat gastrointestinal tract samples.The subculture experiments were carried out 10 times every 24 h,it was found that the E.coli K99-mCherry-CRISPR/Cas constructed by CRISPR/Cas9 technology had the most stable fluorescence expression.Compared with GFP-labeled ETEC,the variation coefficient of mCherry-labeled strains in fluorescence intensity detection was significantly lower(p=0.0328),indicating that mCherry had stronger penetrability and more sensitive detection in all gastrointestinal segments of rats.In conclusion,E.coli K99 was used as a general pathogen to construct a red fluorescent protein-labeled challenge strain.The recombinant strain can not only stably express red fluorescent gene,but also avoid the spontaneous fluorescence interference of animal gastrointestinal tract to achieve sensitivity detection.It provided a methodological basis for the study of specific markers of pathogens and the construction of animal challenge model.