Preparation Of Monoclonal Antibody Against Thioredoxin 2 Of Musca Domestica And Study On Its Anti-oxidation Function

Musca domestica is an insect of the genus Diptera Muscaidae.It has strong reproduction and spreading ability.It is one of the most widely distributed insects in the world known to mankind.Its strong environmental adaptability and anti-adversity defense system have gained more and more attention.The redox system mediates signal transduction and antibacterial immune response by regulating the redox balance during the organism's resistance to pathogens or external environmental stress.Thioredoxin(Trx)is considered to be one of the important antioxidant components of the body.Its antioxidant role has been studied in mammals,but the antioxidant mechanism of insect Trx is less studied.In this experiment,a mitochondrial-specificTrx2(MdTrx2)gene of M.domestica was studied,focusing on the protective function of MdTrx2 on the body and mitochondria under the stress of heavy metals and other oxidative stress inducers.The main research results are as follows:1.According to the transcriptome information,clone the MdTrx2 gene c DNA sequence of720 bp,which contains a complete open reading frame(ORF)of 423 bp,which encodes a protein sequence composed of 140 amino acid residues.The MdTrx2 protein contains Trx domain and typical The redox active site"C-X-X-C"composed of two cysteines.It has 43.08%sequence similarity with humanTrx2 protein.A total of 13 Trx genes were searched in the M.domestica genome,among which the protein encoded by MdTrx2 was predicted to be located on the mitochondria.2.Recombinant expression of MdTrx2 was carried out using the prokaryotic expression system.In vitro experiments showed that the purified r MdTrx2 had reducing activity on the disulfide bond of insulin.Compared with the empty-transformed strain,the recombinant strain of r MdTrx2 showed a significantly higher tolerance to phenazine methosulfate(PMS),confirming that MdTrx2 is involved in the antioxidant process of cells.At the same time,the thermodynamic and kinetic parameters of the binding reaction between r MdTrx2 and heavy metals(Cd²⁺and Cu²⁺)were determined by isothermal titration calorimetry(ITC).The results show that r MdTrx2 has the ability to bind heavy metals Cd²⁺and Cu²⁺.The results indicate that MdTrx2 may play an important function in resist heavy metal stress.3.Use monoclonal antibody technology to prepare MdTrx2 monoclonal antibodies,screen and purify to obtain 4 excellent antibodies.Using the prepared monoclonal antibody,Western Blot was used to detect the protein expression of MdTrx2 in different developmental stages and different tissues of M.domestica.The results showed that MdTrx2 was not expressed in the eggs and pupae of M.domestica,and the expression level of adult flies on the 5th day after eclosion.The expression level has obvious developmental correlation;MdTrx2 is expressed in different tissues of larvae and adult flies,and the expression level is higher in the intestine and fat body of M.domestica larvae,and the malpighian tubule and nerve of adult flies.4.The expression changes of M.domestica MdTrx2 after exposure to doxorubicin(DOX),CdCl₂ and CuCl₂ were detected by fluorescence quantitative PCR from the mRNA level.The results showed that the expression of MdTrx2 increased significantly after treatment with DOX,CdCl₂ and CuCl₂.It shows that these conditional stimuli can promote the transcriptional expression of MdTrx2 gene.5.In order to further analyze the mechanism of MdTrx2 in the process of resistance to heavy metal stress,the expression of MdTrx2 in M.domestica larvae was successfully knocked down by dsRNA microinjection.The larvae were treated with heavy metal Cd²⁺after dsRNA injection.Compared with the control group,the survival ability of the MdTrx2 interference group was significantly reduced,and the activities of mitochondrial complex I and II were also significantly inhibited.It is speculated that MdTrx2 plays an important role in maintaining the mitochondrial redox balance.Under stress conditions such as heavy metals,the loss of MdTrx2function will increase the level of oxidative stress,which in turn will cause damage to mitochondrial function and reduce the activity of complexes I and II.In conclusion,this paper confirms that M.domestica MdTrx2 plays an important role in maintaining the stability of mitochondrial function,participates in the host's resistance to heavy metal stress,and maintains the body's redox balance.