Team:UCSC/Results
Overview
We did site-directed mutagenesis to substitute our proteins containing an N-terminal FLAG-tag with a 6xHis-tag and expressed the new his-tagged proteins CAHS1(D4),CAHS2(D3), LEA1(A4), SAHS10(G3) successfully. We performed virus propagation through embryonated chicken eggs and extracted allantoic fluid containing the live, lentogenic NDV LaSota strain. We also isolated red blood cells (RBCs) from whole turkey blood successfully and ran the hemagglutination assay on our vaccine and allantoic fluid to verify that our NDV vaccine was indeed alive.
Flag Tag Protein Production
During the first phase of our project, we performed a large scale protein production and purification of native CAHS D. We also conducted large scale production of CAHS2 (D3), CAHS1 (D4), SAHS10(G3), LEA1(A4), and rvLEAM(H4); all five of which contained an N-terminal FLAG-tag. After we received a product, we autoclaved them to purify the proteins. The SDS-PAGE gel of the purified products shows that only CAHS D, CAHS1, SAHS 10 and LEA1 had good yield. As a result, we decided to continue site-directed mutagenesis with these four proteins.
Figure: SDS-PAGE gel of purified protein from large scale production. CAHS D, CAHS 1, SAHS 10, and LEA1 have a significant band corresponding to the size of the protein produced.
Since the first step in safety is minimizing the exposure to risk, our team worked hard to eliminate risk wherever possible. We had access to a dry lab space separate from the lab where we could work on various aspects of our project that did not involve direct experimentation. This allowed us to maintain a more controlled lab space. Our team also limited the use of hazardous reagents or equipment when we had access to safer alternatives. For example, we used SYBR Safe gel stain instead of ethidium bromide so that our imaging could be done without UV.
Site Directed Mutagenesis
The reason we did site directed mutagenesis was to remove the FLAG tag with the His tag so that we could use the His column to purify the protein after we had finished producing it. To do this, we needed to use the NEB base changer to design primers that would replace the FLAG tag with an N-terminal 6xHis tag. SDM was then performed using a Q5 polymerase system as well as touchdown PCR based on the manufacturer protocol. Our products were ran in gels as well as sequenced to see the success of the procedure and if the His tag was implemented. Upon confirmation of the procedure, we then transformed the plasmids into dh5-α cells to have stocks of it.
Figure. Gel of results of Site Directed Mutagenesis of CAHS1(D4),CAHS2(D3), LEA1(A4),rvLEAM(H4), SAHS10(G3) This gel shows the results of site directed mutagenesis and the removal of the FLAG tag with a His tag. The presence of bands in the gel indicates that the site-directed mutagenesis was successful.
Protein Production and Purification
Protein Purification after Site Directed Mutagensis
For weeks we were trying to perform protein production with our plasmids and continued failing in producing any. We tried troubleshooting it in multiple ways but still received the same results. Upon looking at the actual structure of the sequence, we realized that we had created a hairpin terminator, which would not allow any protein to be translated.
We ran the pilot protein production of His-tagged CAHSD, CAHS1, LEA1, and SAHS 10 to verify if they were able to be expressed or not. After we verified that they got expressed from pilot expression, we proceeded to large scale protein production and purified the product through autoclaving. The SDS-PAGE gel of the purified large scale proteins indicated there were some other unknown proteins left over after autoclaving, so we did the his-column purification next for further purify.
Figures SDS-PAGE gel of purified 6xHis-tagged CAHS D from pilot expression. Bands in red boxes indicated we got purified protein in heat-soluble fraction.
Figures SDS-PAGE gel of purified 6xHis-tagged CAHS1 from pilot expression. Bands in red boxes indicated we got purified protein in heat-soluble fraction.
Figure. SDS-PAGE gel of purified 6xHis-tagged SAHS 10 from pilot expression. Bands in red boxes indicated that we got purified SAHS10 in heat-soluble fraction.
Figure. SDS-PAGE gel of purified 6xHis-tagged CAHS1(D4), CAHSD, LEA1(A4), and SAHS10(G3) from large scale production. Bands in red boxes indicated we got purified proteins in heat-soluble fraction.
HIS Column Purification
After we purified proteins by autoclaving the large scale product, we ran the his-column to further purify the His-tagged proteins. By running through different concentration of imidazole in elution buffer, we found out that 100mM is the best concentration for eluting protein.
Figure. SDS-PAGE gel of His-column purified CAHS D. This image shows successful purification of CAHS D. Based on the results of the gel, we can say our best results occur when the samples were in 100mM elution buffer.
Figure. SDS-PAGE gel of His-column purified CAHS 1. Purified CAHS1 away from DNA and the remaining proteins. The supernatant was not filtered before use, the column was clogged, making purification take over 20 hours. In addition, it appears we overloaded the column as there was still a significant amount of protein that did not bind. We could get better yield after optimized the imidazole concentration in elution buffer.
Figure. SDS-PAGE gel of His-column purified SAHS 10. The image displays successful purification of the protein. The band in the red box indicated that we got the most yield of protein from 100mM imidazole elution buffer.
Hemagglutination Assay
We got the whole Turkey blood and isolated RBCs by following the protocol Isolation of RBCs.
In order to test if we have hemagglutinating agent in the vaccine sample and if the RBCs we isolated before will auto-hemagglutinate, we did a rapid hemagglutination test and looked at the sample under the microscope. The combination of vaccine and the RBCs had clumped appearance which distincted from the control of PBS with RBCs after mixing about one minute. However, after 10 minutes heat inactivating, the vaccine sample combined with the RBCs had the same appearance with the control of PBS with RBCs. This rapid test indicated that we could use hemagglutination assay to test the infectivity of the NDV-La Sota strain in the future.
Figures: From Left to Right Rapid hemagglutination test under microscope
1.) Control of RBCs in PBS.
2.) 10 minutes heat inactivated vaccine sample with RBCs.
3.) New stock of vaccine with RBCs
After we did the rapid test and determined our isolated RBCs are ready to run the hemagglutination assay, we performed the HA assay with both new stock, old stock vaccine, allantoic fluid from both new and old vaccine stock and the 10^-5 dilution of the new vaccine stock.
Figure. HA of the new and old stock vaccine of different dilution. Both of the new and old stock vaccine have HA titer of 128.
Figures: From Left to Right
Figure. Hemagglutination assay of Allantoic fluid from embryonated eggs after virus propagation. In the left figure, the first two lanes stand for the allantoic fluid from old vaccine stock and the HA titer is 16. The following two lanes stand for the allantoic fluid from new vaccine stock and the HA titer is 32. The next two lanes stand for the allantoic fluid from -5 dilution of new vaccine stock and the HA titer is 8. In the right figure, the first two lanes stand for the allantoic fluid from 10^-3 new vaccine stock and the HA titer is 2. The following two lanes stand for the allantoic fluid from 10^-1 new vaccine stock and the HA titer is 32. The bottom two lanes of both of the figures stand for negative control which was RBCs without virus.
Our first rapid hemagglutination test of the vaccine stock under heating conditions indicated virus death when exposed to heat at 56 °C for 10 minutes. We performed more rapid hemagglutination tests under heat using different times and temperatures. Each test contained one positive control-vaccine sample with RBCs we had previously tested and verified successful hemagglutination and one negative control-PBS with RBCs which we tested to verify the RBCs will not auto-hemagglutinated. The results we looked under microscope indicated that virus under heating at 37.5℃ and 26.6℃ for 10 minutes stayed alive. Virus under heating at 56℃ for 5 minutes still died which indicated that 56℃ is a significant killing temperature to NDV-La Sota strain. We also did a test of virus centrifuged at 3,000 rcf under 37.5℃ and the result showed the virus stayed alive. This indicated that when we dilfiltrated the combination of the virus and proteins through the centrifuge, the virus stayed alive, so our HA assay on them were reliable.
Figure. Duplicated Rapid HA test under different conditions.
Figure. The first two lanes stand for negative control. The next two lanes stand for positive control and the next two lanes stand for 37.5 degree with TDP after 10 minutes. and the last two lanes stand for 37.5 degrees without TDP after 10 minutes.
Figure. First two lanes stand for difiltration with TDP. Didn't have enough. smaller than 50 ul spun without TDP
Plaque Assay
Our plaque assay tests were failed for the first two rounds. Thus, we spent two weeks diagnosing our procedure. We finally found out that the concentration of the agar is the essential problem here. By running the 0.9%, 0.6% and 0.3% agar overlay medium with both 2X PBS and 2X TPB on the same condition of chicken fibroblast cell monolayer, we figured out the 0.3% agar
