| Nowdays, the infection of HIV is a serious disease threaten the health of human beings and stability of society. It is an important method to develop the effective anti-HIV vaccine to lower the morbility rate of infectors and avoid more people to catch HIV.HIV is classified into type 1 and type 2, the HIV-1 is widespread now. HIV-1 Tat(trans-activator of transcription)protein is a key viral regulatory protein and is necessary for viral gene replication,spreading and pathopoiesis.After the target cells infected with HIV, the Tat generated in cytoplasm can reach the nucleus and binds to the transactivating responsive region(TAR) to prolong the HIV-1 new RNA. In addition, Tat is secreted from infected cells into extracellular medium to have the important role of pathopoiesis. As a result, blocking the influence of virus replication and ecto-toxic effect is the mainstay to develop the prophylactic and therapeutic Tat vaccine.Tat protein produced by prokaryotic expression has the more stabile conformation, higher antibody titer and better neutral activity, but its quatity is less, which restrict the large scale production of Tat protein. In contrary, Tat protein produced by chemical synthesis has the advantage of higher purity and more quatity, but its conformation is instability, antibody titer is lower and neutral activity is weaker. So, it has the very important meaning to improve the expression level, increase its stability and remain its immunogenicity.Four parts:1. Expression, purification and immunogenicity analysis of HIV-1 HXB2 subtype Tat(1-101aa) protein in E.coli The primers were designed according to the Tat amino acid sequence of HIV-1 HXB2 subtype and the preferred codons of E.coli. The Tat(1-101aa) DNA fragment was synthesized in vitro by overlapping PCR and sequenced after inserted into pMD-18T vector by T/A method. Then the correct Tat(1-101aa) DNA sequence was coloned into pET32a vector which is used as expression vector. The recombinant plasmid pET32a- Tat(1-101aa) was constructed successfully and transformed into in E.coli BL21(DE3) strains. The pET32a- Tat(1-101aa) was induced by IPTG and the relative molecular weight(MWw) of expressed product was 31 000. Purifing the expressed product by the Ni-NTA column and the dense of the purified PET32a-Tat(1-101aa) protein was 1.50mg/ml. Three rabbits were vaccinated with PET32a- Tat(1-101aa) protein, then testify the reactivity of sera from rabbits evaluated by ELISA. The result testified the antibody titre raised by PET32a- Tat(1-101aa) protein was 640 000 and the anti-sera of PET32a- Tat(1-101aa) reacted specifically with Tat(1-101aa) protein.2. Segmentative expression, purification and immunogenicity analysis of HIV-1 HXB2 subtype Tat protein in E.coliTo discover the region which affect the prokaryotic expression of HIV-1 HXB2 subtype Tat protein and its important epitopes , we divided the Tat protein into three regions(1-48 amino acids, 22-72 amino acids and 38-101 amino acids).Tat(1-48aa),Tat(22-72aa)and Tat(38-101aa)DNA fragments were obtained in vitro by PCR and cloned into pMD-18T vector by T/A method. Then the correct Tat(1-48aa),Ta(t22-72aa)and Ta(t38-101aa) DNA sequence were coloned into pET32a vector which is used as expression vector. The recombinant plasmid pET32a-Tat(1-48aa),pET32a-Tat(22-72aa)and pET32a-Tat(38-101aa)plasmids were constructed successfully and transformed into E.coli BL21(DE3) strains. They were induced by IPTG and the relative molecular weight(MWw) of expressed products were 23 000, 23 000 and 25 000. Purifing the expressed products by the Ni-NTA column and the dense of the purified PET32a-Ta(t1-48aa),PET32a-Ta(t22-72aa)and PET32a-Tat(38-101aa)protein were 4.26mg/ml , 3.35 mg/ml and 5.11mg/ml. ELISA testified the anti-sera of PET32a-Tat(1-101aa) protein from rabbtis can reacted specifically with PET32a-Tat(1-48aa),PET32a-Tat(22-72aa)and PET32a-Tat(38-101aa)protein, the titres were 1:128 000, 1:32 000, 1:64 000 respectively and its reaction with PET32a protein was weak. The results demonstrated cysteine-rich region(22-37aa) can affect the expression of Tat protein greatly, the N-region, basic region , C-region are the important epitopes of Tat and N-region is the most significant one of them.3. Effective expression, purification and immunogenicity analysis for HIV-1 HXB2 Subtype Tat Protein deleted the cysteine-rich region in E.coliDeleting the cystein-rich region (22-37 amino acids)of HIV-1 HXB2 Tat protein(whole length is 101 amino acids) to improve its expression level and stability in E.coli and develop the novel antigen for HIV Tat vaccine.Tat DNA which deleted the cysteine-rich region (64-111 nucleotides), named as Tat(△C)DNA, was obtained in vitro by PCR and cloned into pMD-18T vector by T/A method. Then the correct Tat(△C) DNA sequence were coloned into pET32a vector which is used as expression vector. pET32a-Tat (△C ) plasmid and the pET32a-Tat(1-101aa) plasmid were established and transformed into E.coli BL21(DE3) strains respectively to conduct the protein expression and purification at the same conditions. They were induced by IPTG and the relative molecular weight(MWw) of expressed products were 29 000 and 31 000. Purifing the expressed products by the Ni-NTA column and the dense of the purified PET32a-Tat(△C)protein was 7.12mg/ml,which was greatly more than PET32a-Tat(1-101aa) protein(1.50mg/ml).Dimer of PET32a-Tat(1-101aa) protein can be observed just after the protein purification and stored at 25℃and 4℃for 7 days, but dimer of PET32a-Tat(△C)protein was not formed at the same condition. The stability of Tat protein deleted the cysteine-rich region can be increased greatly.Three rabbits were vaccinated with PET32a-Tat(△C )protein,then testify the reactivity of sera from rabbits evaluated by ELISA and Western-blot. Experimental rabbits were immunized with PET32a-Tat(△C)protein and produced high titre of anti- PET32a-Tat(△C)serum(1:320 000),the antibody can react specifically with Tat(△C)protein, Tat protein (1-101aa)and synthetic Tat(1-86aa) protein.4. The comparision for the reactivity of sera from HIV-1 infectors with different Tat antigen.Detecting the reactivity of sera from HIV-1 infectors with prokaryotic expressed PET32a-Tat(1-101aa),PET32a-Tat(△C )protein and synthetic Tat(1-86aa) protein to compare their positive ratio and specificity of the reactivity. The aim is for selecting the best antigen to be used in the clinical detection.The positive ratio of PET32a-Tat(△C )protein was 13.5%, which was greatly higher than the other two antigens. The A450 average value of the positive reactivity of sera from HIV-1 infectors with PET32a-Tat(△C)protein was 0.193, which was higher than the other two antigens. The A450 average value of the negative reactivity of sera from HIV-1 infectors with PET32a-Tat(△C) protein was 0.024, which was lower than prokaryotic expressed PET32a-Tat(1-101aa)protein. The results demonstrate PET32a-Tat(△C)protein has the strongest reactivity with the sera from HIV-1 infectors and deleting the the cystein-rich region can decrease the non-special reactivity with the sera from HIV-1 infectors.We discover the the region which affect the prokaryotic expression of HIV-1 HXB2 subtype Tat protein――cystein-rich region and construct the pET32a-Tat(△C)plasmid successfully. The expression level in E.coli and the stability of Tat protein deleted the cysteine-rich region can be increased greatly, the protein remains good immunogenicity and obtains the cross-reactivity. PET32a-Tat(△C )protein has the strongest reactivity with the sera from HIV-1 infectors and deleting the the cystein-rich region can decrease the non-special reactivity with the sera from HIV-1 infectors. The results provide not only a probable novel antigen for further development of HIV Tat vaccine, but also a new pattern for the construction and function study of Tat protein.
|
PDF Full Text Request