Construction Of Artificially Designed Camelid Single Domain Antibody Library And Anti-estradiol VHH Antibody Screening

Food safety is a hot problem for the public health all over the world. It was reported that the food safety problem is most concerned in Chinese; surpass other problems of public safety, such as transportation safety, health safety and environment safety. Strengthening the food hazard factors detection is an effective measure for solving the food safety problems. For the immunological analysis techniques of food safety detection, rapid producing specific antibody of clear background is one of the most important works.This research was based on the camelid single domain antibody; physical library and virtual library were constructed by gene sequencing and computational biology technologies respectively. The typical estrogen such as estradiol was selected as the target; the antibody screening platform was preliminary validated by screening the estradiol-specific single domain antibody, binding activity, competitive activity and affinity of the selected antibody was analyzed by ELLISA and SPR. The camelid single domain antibody library was successfully constructed by computational biology techniques and the anti-estradiol antibody was selected. 1. Construction of single domain antibody gene library employing first generation of sequencing techniqueFirst of all, peripheral blood was taken from the external jugular vein of healthy adult Bactrian camel, peripheral blood mononuclear cell was extracted and the method of TRIZOL was applied to extract the total mRNA. Then the first-strand of cDNA synthsis kit was used to acquire the cDNA library. By well-designed primers, the VHH gene segments were amplified from the cDNA library. Using TA cloning technology, the recycled VHH genes were cloned into the pMD18-T plasmid. Through the analysis of sequencing results, the VH and VHH genes were distinguished by identifying the residues of 37, 44, 45 and 47 sites in the FR2, and the VH gene segments were deleted. Finally the construction of VHH gene library with about 2000 variants of VHH antibody was built.In our experiments, the VHH gene segments were acquired from healthy adult Bactrian camel through first generation of sequencing technology. The VH genes were picked by identifying the conserved residues in FR2. Then the homology analysis was performed to found the identical VHH genes. Finally, their DNA and amino acid sequences were clear through the form of DH5a-pMD18-T-VHH, the VHH antibody in the gene library 2. Construction of VHH virtual library and optimization of the key parameters for antibody models in the computational biological platform.Based on the construction of VHH gene library, the homology modeling method and molecular dynamics simulation were applied to build the VHH virtual library. First, the amino acid sequence of every VHH was submitted to the website of Abnum for antibody numbering, and then, which the CDR and FR could be identified. After antibody numbering, the amino acid sequences of VHH were submitted to the website of SWISS-MODEL for homology modeling. The homology modeling method belongs to the comparative modeling, and it is the most reliable method in protein structure predicting. To modify the loops of VHH, molecular dynamics simulation were applied to every model of VHH. The supercomputer in the National Supercomputer centre was used. The GROMACS software package was used to perform the MD simulation. The energy minimization, temperature equilibration and pressure equilibration were finished; the time of molecular dynamics simulation was set 10 ns. Finally, the RMSD and Gyrate were analyzed to assess and selected the modified model of VHH.In this experiment, the FR and CDR for every VHH antibody were identified. Generally, the four FR parts are folded into the scaffold of antibody, and the structures of three CDR parts are random loops that are responsible for the antigen recognition. The loop structures of CDR showed great diversities, which may relate to the nonconservative protein primary sructure of antibody and the flexibility of the loop structure. So the molecular dynamics simulation was performed to modify the structure of VHH. During the simulation process, the stored energy due to the improper geometry and steric hindrance was released, and its temperature and pressure were also balanced, the state of the whole system was subsequently guaranteed. The RMSD and Gyrate was analyzed after the simulation. The structure of VHH antibody was found to be stabilized and the protein folding of VHH became compact. After the above procedures, the modified models for 2000 strains of VHH in the library were acquired, and the different domain for every antibody structures was clearly distinguished, which was helpful for the docking analysis.In total, the single domain antibody virtual library was constructed in which every VHH antibody strain has its own clear protein secondary and tertiary structures, and the antigen-binding domain was also well optimized for subsequent virtual screening experiments. 3. Virtual screening of specific VHH antibody on representative estradiol targetTo preliminary validate the VHH antibody screening library, estradiol, as the target which one of the estrogen veterinary drugs residu exist animal food such as meat, egg and milk was chosen. For the virtual screening strategy, molecular docking was used to perform the virtual screening of anti-estradiol VHH antibody from the VHH library. Among the docking softwares, Auto Dock package is being used most frequently. In this research, the space around the three CDR loops were selected as the estradiol binding domain of VHH, the residues located in the three CDR were set to be flexible. By running the AutoGrid programme, different kinds of atom probe were used to compute the grid energy. When analyzing the docking results, the specific VHH antibodies were picked according to the binding affinity, and the binding modes of estradiol-VHH were also analyzed.By analyzing the anti-E2 VHH, three loops of CDRs were found to form the proper antigen-binding domain to provide the favorable spatial conformation for binding estradiol. For the binding mode analysis, the element of nitrogen of the amino acid residues located in the CDRs, especially the CDR3, involved in the formation of H-bond with the hydroxyl hydrogen of the estradiol. The aromatic ring of the amino acid could also interact with the benzene ring of estradiol via π-π interaction. In total, the docking analysis was performed with molecular docking technology, and the virtual screening strategy of the VHH virtual library was realized. Besides, the supercomputer was used to efficiently complete the screening of 2000 VHH antibody in the library.The method of molecular docking was chosen to perform the virtual screening for the VHH antibody screening platform in this research. Compared with the conventional antibody screening method, the selected specific antibody from the virtual library could be effiently acquired, the informations of binding mode of antigen-antibody complexes could also obtain. Meanwhile the analysis results would provide detailed knowledge for subsequent antibody design and affinity maturation. 4. Expression, purification of VHH and properties analysis of anti-E2 VHHSeries of pET plasmid was chosen for the expression of VHH. First of all, the forward primer and downward primer with designed BamHI and Eco RI restriction sites respectively were used to amplify the VHH genes. To ensure the effect of enzyme digestion, the VHH gene segments were primarily cloned into the pMD18-T plasmid, then double enzyme digestion were applied to the pMD18-T-VHH and pET32 a. The digested VHH and linear pET32 a were recombinate using T4 ligase. After the pET32a-VHH being transformed by BL21(DE3) competent cell, the positive clones of BL21(DE3)-pET32a-VHH were characterized by colony PCR. The sequences of positive VHH were analyzed after gene sequencing, and the ORF of VHH antibodies were also examined. During the expression of VHH, the BL21(DE3)-pET32a-VHH was activated firstly, then the activated strain was transplanted to the big medium and cultivate under 37℃ until the OD600 of culture reached about 0.5-0.6, IPTG was added to induce the expression of VHH under 37℃ for about 12-15 h overnight. By performing the SDS-PAGE for analysis, the amount of VHH protein was found to be acquired with high-level. After the cell disruption using ultrasonic, the VHH protein formed into inclusion body. The the renaturation assa y was performed to get the soluble VHH antibody, which was quantitatively determined by BCA kit.In the properties analyzing assays, the antibody VHH165 with lowest binding free energy was selected for the validation. The binding property, competitive property and binding affinity were analyzed with indirect ELISA and SPR. The OD value between VHH165 and E2-OVA increased with the increasing of the concentration of VHH165, and was higher than that between VHH165 and OVA. Through indirect ELISA, the 50% inhibition concentration(IC50) for E2 was 20 ng/mL, and the limit of detection(LOD; IC10) was 4.097 ng/mL. While VHH165 showed acceptable affinity to E2 with KD of 1.49 μM detected by SPR.In this study, the combination of first generation of sequencing and computational technology was used to construct the camelid single domain antibody library, which including physical and virtual library. And molecular docking was applied for rapid virtual screening of anti-estradiol VHH antibody. By analyzing the binding mode of E2-VHH complex, the binding information was also acquired. An effient expression system of camelid single domain antibody had been established. The VHH antibody screening platform and virtual screening strategy were preliminary validated by determing the binding activity, competitive property and binding affinity. There are two major advantages of this VHH antibody screening platform compared with the antibody screening method established by conventional methods: on one hand, this antibody library was based on the camelid VHH antibody, this kind of miniaturized antigen-binding segment has low molecular weight, and it is thermostable, soluble, resilient to environmental factor, easy to be genetic manipulation and preserved; on the other hand, with the aid of computational biology technology, it is clear in the aspects of genetic background, protein secondary, tertiary structure, as well as the binding mode of target-antibody complexes. In the future, based on the above information, the specific antibody from this antibody library could be virtually designed. By building the physical and virtual naive camelid VHH antibody library, the subsequent affinity maturated specific antibody would be prepared in a rapid and informationalized mode.After the construction of single domain antibody library, the estradiol specific VHH antibody with good affinity was acquired. Meanwhile, the selected anti-E2 VHH had comprehensive biological information, by which the further affinity maturation and antibody virtual design would be rapidly realized in silico.