The Preparation And Characterization Of The Nanobody Against Thioredoxin Glutathione Reductase Of S. Japonicum (Sj TGR)

Schistosomiasis is a zoonotic parasitic disease caused by infection and parasitizing ofSchistosoma japonicum, which is severely harmful to human health. At present, morethan230million people are infected with schistosome in74countries globally.Schistosomiasis epidemic exists in12provinces, a total of452counties, in China.Schistosoma japonicum infection can cause severe pathological changes of humanorgans (including liver, intestinal and urethra canal, etc.), such as the liver egggranulomatous inflammation, cirrhosis, ascites and hepatic coma. Schistosome has acomplex life cycle, and its surface antigen components vary dramatically in differentlife stages. These surface antigens will regulate the host immune system actively,which contributes to immune evasion, and the great difficulties to the development ofvaccines against schistosomiasis. Up to now, no effective schistosomiasis vaccine isavailable, and the schistosomiasis control is still mainly relying on chemical drugs. Atpresent, praziquantel is the only effective and globally used drug for schistosomiasis.The risk of praziquental resistance is rising due to the repeated administration ofpraziquental in some hyperendemic areas, and the development of novel effectivemedicines is in urgent need.Antibodies have been widely used in the scientific and industrial fields, as wellshowed a great application prospect in the biomedical field. The monoclonal,polyclonal and engineered antibodies against target molecules of pathogen, tissue,tumor specific antigen or tumor associated antigens, cytokines and their receptors,hormone and some oncogene products, which are prepared by the traditional immunemethod or cell engineering, genetic engineering technique now have been widelyused in disease diagnosis, treatment, immune intervention, and scientific research. Invertebrates, antibodies are the natural barrier of preventing organs from the invasionof pathogens, malignant cells and toxic molecules by binding specifically to targetmolecules. Therefore, it is a very reasonable choice of using antibodies as therapeuticagents or a targeting carrier. However, the mouse derived monoclonal antibodies can cause serious human anti mouse antibody responses (HAMA) in the clinical treatment,and with the disadvantages of large molecular weight, poor penetration ability, strongimmunogenicity and poor stability, which make a huge hindrance in the antibodyapplication. Therefore, preparing the novel antibody of lower molecule weight, lowerimmunogenicity, easy production and storage is very important for improving theapplication value of antibody. There is a kind of functional heavy chain antibody IgG(HCAb) in camel serum, which is naturally lack of the light chain of the classicantibody, the function of the variable region (VHH) of HCAb is similar to thevariable domain of the heavy chain of the classic antibody, and it is also called as thesingle domain antibody or nanobody. The advantage of nanobody include: withsmaller molecule weight (about1/10of the ordinary antibody), it’s easier to entertissue and cell; with a longer CDR3region, which forming a special large convex ringstructure, and easy penetrating into the crevice or groove of the enzyme activitycenter to inhibit the proteinase activity strongly. In addition, nanobody is moresoluble, could be expressed and produced in the prokaryotic cells; with a goodstability, and could tolerate the worse environment of hot, acid and alkali; with alower immunogenicity and is not prone to cause rejection, etc. All of these excellentcharacters make the nanobody potent as the novel therapeutic antibody.In recent years, the redox metabolism balance has gradually become an importanttarget for developing new drugs. Schistosome parasitizes in the mesenteric vein of thehost, in order to keep its normal life activities, it is necessary for them to maintain theredox balance of them as well as resist the injury of reactive oxygen derived from itshost. Researches performed in China as well as other countries have confirmed thatschistosome depends solely on one multi function enzyme-thioredoxin glutathionereductase (TGR) to maintain the redox balance, and inhibiting the function of thisenzyme will lead the death of schistosome, which convinced that TGR is a potentialtarget for developing novel agents against schistosome infection. Therefore,developing chemical inhibitors or antibody against the enzyme activity of Sj TGRwill be of great significance for developing novel drugs of treating schistosomiasis,and the specific antibody against Sj TGR will also be used as a targeting carrier ofchemotherapeutic agents against schistosomiasis.Up to now, there is no report on the research of nanobody against Sj TGR, in thisstudy, a nanobody phage display library against Sj TGR was constructed, therecombinant phages displayed the nanobodies against Sj TGR were screened by bio-panning method, the genes encoding the nanobodies were identified andnanobodies against Sj TGR were prepared and purified by the prokaryotic expressionmethod. This work laid the foundation for the development of new therapeutic agentsor methods against schistosomiasis. The main research works as the following:Part I Construction and identification of the nanobody phage display libraryagainst Sj TGRA female Xinjiang Bactrian camel was immunized with500μg Sj TGR protein byintracutaneous injection in the neck skin. The complete Ferund adjuvant antigen wasused in the first immunization, the incomplete adjuvant antigen was used in the roostimmunization, and the immune interval was two weeks. At two weeks post the lastimmunization，the titer of antibody IgG against Sj TGR of camel serum reached1:12800, which indicated the immunization was successful. Then the leukocytes ofperipheral blood of the immunized camel were isolated and used to prepare the totalRNA and mRNA, the first strand cDNA was synthesized by reverse transcriptiontechnique. The gene specific primer of camel nanobody was designed and synthesizedaccording to the published gene data of the two kinds of subtypes of heavy chainantibody IgG2and IgG3. The DNA fragment of encoding nanobodies were amplifiedby PCR using the first strand cDNA of immunized camel leukocytes as template, thelength of PCR products were about450-500bp, which is consistant to the publishedsize of the variable region gene of IgG2and IgG3. All the nanobody gene DNAfragments were subcloned into the phagemid pCANTab5E via Sfi1/Not1restrictionenzyme sites, and the ligation products were transformed into competent cells ofE.coli TG1to construct the phagemid library, the titer of the library was4.5×1010CFU/mL. The phagemids of20single colonies selected randomly were extracted anddigested by restrictive enzyme Sfi1/Not1, the electrophoresis results of the digestedproducts showed that the recombinant rate of phagemid library was100percent,which indicated that this phagemid library is a high-quality nanobody gene library.Part II Screening and identification of the recombinant phages displayed theXinjiang Bactrian camel nanobody against Sj TGRThe bacteria of nanobody phagemid library were infected with the helper phageM13KO7to package the phagemid DNA of bacterium into the intact phage, afterreproducing, the recombinant phages were released from bacterium (This process wascalled as phage rescue), a4×1012pfu/mL of phage display library was obtained and the library volume was8mL. The recombinant Sj TGR protein was coated on the12wells cell culture plate, and reacted with the recombinant phages of the nanobodyphage display library to pan the recombinant phages displayed the specific nanobodyagainst Sj TGR. Three round panning were performed, the amount of the coated SjTGR was declined from1000μg to10μg, and the concentration of Tween-20in theelution buffer was increased from0.05%to0.5%. After3round panning, therecovered phages of the third round panning increased about1000times than that ofthe first round panning, and target phages were enriched significantly. The phagesfrom third round panning were used to infect the bacterium TG1, and10singlecolonies were selected randomly to extract phagemid, which were digested by Sfi1/Not1enzyme, the electrophoresis results of the digested products showed that80percent of the eluted phages in third round panning contained the inserted foreigngene. The phagemid of100single colonies were sequenced and93sequencing weresuccessful, among them,69phagemids contained inserted foreign DNA fragments ofabout500bp. The inserted exogenous DNA sequences were translated into aminoacid sequences, and were used to amino acid sequence aligned,3kinds of repeatedsequences were found among69amino acid sequences, which were named as VHH3,VHH54and VHH67. The gene encoding the nanobody VHH3is429bp, andpresented27times repeatedly; the gene encoding the nanobody VHH67was441bp,and presented8times repeatedly; the gene encoding the anybody VHH54was441bp,and presented2times repeatedly; other nanobodies presented solely. All of thesethree nanobodies contained two hallmark cysteine residues of heavy chain variableregion antibody, and contained the characteristic hydrophilic amino acid loci F37,E44, R45, and G47in their framework region2, meanwhile its C terminal containedthe GTNEVCK domain, which is the specific hinge sequence of IgG3subtype. Allabove characterization indicated that these3nanobodies belong to the IgG3subtype.Part III The prokaryotic expression and function analysis of XinjiangBactrian camel anti Sj TGR nanobodyThree nanobody genes were subcloned into the expression vector pET28a（+） toform the recombinant expression plasmid VHH3-pET28a, VHH54-pET28a andVHH67/pET28a. Then, the recombinant expression plasmids were transformed intoexpression host BL21（DE3）respectively, and induced with isopropyl-β-D-thiogalactopyranoside (IPTG). The transformants could express a recombinant nanobodyof about20kDa. These recombinant nanobodies were proved to be derived from the camel, because which could be recognized by the rabbit anti camel IgG secondantibody specifically. The recombinant nanobodies were purified from the expressionproducts through the Ni-NTA nickel ion metal affinity chromatographic method. Theimmunoblotting experiments confirmed that all the recombinant nanobody VHH3,VHH54and VHH67protein could bind with the recombinant Sj TGR protein, and thenanobody VHH3could inhibit the activity of thioredoxin reductase (TR) of Sj TGReffectively, but the nanobody VHH54and VHH67did not inhibit the TR activity ofSj TGR, which suggested that the nanobody VHH3could binding to the regionrelated to the activity center of Sj TGR molecule, but the binding sites of thenanobody VHH54and VHH63did not relate to the activity center of Sj TGR.Conclusion: In this study, we successfully constructed a large capacity of anti Sj TGRXinjiang Bactrian camel nanobody phage display library, three recombinant phagesdisplayed the nanobody against Sj TGR specifically were obtained through the solidpanning. Three recombinant nanobodies against Sj TGR were prepared byprokaryotic expression method, and the purified recombinant nanobody VHH3caninhibit the TR activity of Sj TGR effectively. These works laid a good foundation fordeveloping a valuable nanobody against Sj TGR for treatment and targeting carrier infuture.