Construction And Identification Of Toxoplasma Gindii SRS29C Gene Knockout Strain

Toxoplasma gondii is an obligate intracellular parasitic protozoa that can infect all warm-blooded animals including humans.It is an important zoonotic parasite with worldwide distribution.Toxoplasma gondii is a typical opportunistic pathogenic parasite and does not show obvious clinical symptoms when it infects healthy hosts.But when it infects immunocompromised hosts,it can cause severe toxoplasmosis and even death.In addition,toxoplasmosis infection in pregnant women can cause miscarriage and stillbirth,and can cause congenital toxoplasmosis in developing fetuses.The average infection rate of toxoplasmosis in China is 10%.Toxoplasmosis infection in domestic animals is mainly manifested as abortion,stillbirth,mummified fetus,etc.,and the quality of meat and dairy products is reduced.Toxoplasmosis has brought huge economic loss and potential threat to the breeding industry and human public health.The invasion process of Toxoplasma gondii is complex.Understanding the invasion process of Toxoplasma gondii into the host is helpful for the prevention and treatment of toxoplasmosis and the research of reliable vaccine.The surface proteins of Toxoplasma gondii play an important role in the invasion of host cells.In this paper,a surface antigen protein SRS29C was studied.（1）The recombinant plasmid SRS29C-o GEX-4T-1 was constructed by escherichia coli prokaryotic expression system and transformed into Transetta and BL21.The recombinant protein SRS29C-GST was successfully expressed in both of them.Since the protein is expressed in the form of insoluble inclusion body,the obtained protein is purified by inclusion body washing.The purity of the recombinant protein after inclusion body washing met the test requirements.Then,New Zealand white rabbits were immunized with the recombinant protein to prepare polyclonal antibody against rabbit SRS29C fusion protein.ELISA and Western-blot tests showed that the highest anti-polyclonal antibody titer was 1:320 000 and the immunogenicity was good.IFA assay was performed using the prepared antibody and SAG1 antibody expressed on the surface of T.gondii.The results showed that SRS29C was expressed on T.gondii surface.（2）The SRS29C gene knockout guide plasmid,homologous template plasmid,fixed-point replenishment guide plasmid and fixed-point replenishment overexpression plasmid required by CRISPR/Cas9 technology were constructed by point mutation technique and seamless cloning technique,which all met the test requirements after sequencing.The SRS29C gene knockout strain of Toxoplasma gondii was constructed by electrical transformation.PCR verification was conducted using the designed primers for gene knockout specificity detection,and the positive SRS29C gene knockout strain was screened out.At the same time,the prepared polyclonal antibody was used to detect the expression of SRS29C protein by Western-blot.PCR identification showed that 797 bp 5’UTR（SRS29C）fragment and 576bp3’UTR（SRS29C）fragment could be amplified,but no SRS29C fragment could be amplified,while 700 bpSRS29C fragment could be amplified in the control group.And SRS29C protein was not expressed in the protein verification gene knockout strain.These results indicate that the SRS29C gene knockout monoclonal strain of T.gondii was successfully constructed and screened out,which lays a foundation for the functional study of T.gondii SRS29C gene.