Establishment Of Chromosome-Plasmid Balance Lethal System Of Expression Of 3-1E Gene Of Eimeria Tenella

Chicken coccidiosis caused by Eimeria which has a high rate of the disease that influence the aviculture industry.So far,the main method of controlling coccidiosis is drugs and live vaccines.However,due to prolonged use of drugs and increased vaccine costs,there is an urgent need to find a more effective and safer alternative strategy to control coccidiosis.The method of delivering Lactic acid bacteria as an antigen delivery system is a safe way.This article explores the establishment of the non-resistance selection markers of Lactic acid bacteria expression system,and has established the expression system of the 3-1E which was relatively conservative in E.tenella.E.acervulina and E.maxima.Firstly,we design two primers of the thy A,and regard Lactococcus lactis NZ9000 genomic DNA as a template,aquiring upstream and downstream segments of thy A and then cloned into PMD18-T cloning vector,the plasmid identified as positive by the above steps was sequenced.thy A-up-F/R、thy A-down-F/R as primers and acquiring the two thyA segments and then cloned into p Ghost9 to construct the p Gthy A-up-down plasmid,which 861 bp base of thy A has lost.The vector was electroconductive into Lactococcus.lactis.subsp.cremoris NZ9000,according to the principle of homologous recombination,we acquired single crossed colonies and double crossed colonies.Acquiring double crossed colonies genomic DNA and L.lactis NZ9000 by PCR and blast,we find that we has 861 bp lost.The results show that we have acquired auxotrophy strain NZ9000/ΔthyA。The promoter and ribosome binding site sequence of ThyA gene of L.acidophilus strain were predicted by software.Design thy A gene primers containing P_LAB regions.Acquiring the thyA target gene fragment and then cloned into PMD18-T cloning vector,the plasmid identified as positive by the above steps was sequenced.The steps identified as positive for the plasmid as a template,P_LAB-Thy ALAB as primers and acquiring the P_LAB-thy ALAB purpose fragment.And then cloned into p TX-8048-3-1E,the pasmid was electroconductive into NZ9000/Δthy A.Identification of positive transformants by PCR and digestion.The correct strains were tested on SA agar plates（without thymidine）to verify P_LAB expression.A single colony of platelets was picked up into BL liquid medium for 20 passages.Plasmids were extracted and the plasmids were tested for stability.The obtained strain was expressed by western-blot.The results show that the plasmid p TX-8048-3-1E-P_LAB-thy ALAB was successfully constructed and the P_LAB gene was successfully expressed in NZ9000/ΔthyA-p TX-8048-3-1E-P_LAB-thy ALAB strain.After 20 passages,the plasmid was stable and the E.tenella 3-1E protein was successfully expressed in the non-resistant vector.A pair of reverse PCR primers Remove-Cm were designed,p TX-8048-3-1E-P_LAB-thy ALAB as template and the purpose fragment was acquired by PCR.The target fragment was self-ligated by Not I restriction site,and the Lactic acid bacteria expression vector p TX-8048-3-1EP_LAB-thy ALAB（Cm removed）was obtained.Electroconduct it into NZ9000/Δthy A,and the Lactobacillus expression vector NZ9000/ΔthyA-p TX-8048-3-1E-P_LAB-thy ALAB（Cm removed）was obtained by screening the non-resistant selection marker.The expression of the target protein E.tenella 3-1E was detected by western-blot,and the strain was passaged for 20 generations.After identification is correct,the expression of target protein E.tenella 3-1E was detected by western-blot.The results showed that the expression vector of non-resistant selection marker was successfully constructed.The expression of E.tenella 3-1E protein was successfully expressed in the non-resistant vector.The culture medium was continuously passaged for 20 generations,and no plasmid was found to be lost and the plasmid was stable.And the E.tenella 3-1E protein is still expressed.In conclusion,the thy A auxotrophic strain NZ9000/Δthy A was successfully constructed and a non-resistant selection marker vector was constructed with NZ9000/Δthy A as the recipient strain,and the chicken E.acervulina surface antigen 3-1E was successfully expressed.