【文章內(nèi)容簡(jiǎn)介】 epitope we have maintained the Tat core region 3848aa. The Tat basic region of 3861 fragment was built into the prokaryotic expression vector, then we purified Tat (3861) fusion protein and tested their immunogenicity in order to analyze whether this important epitope is retained.The primers were designed according to the Tat sequence of HIV1 strain and the preferred codons of . The tat(3861) were synthesized in vitro by PCR and sequenced after inserted into pMD18T vector by T/A cloning. Then the correct tat(3861) was inserted into pET32a vector to construct prokaryotic expression plasmids pET32atat(3861) and . The rebinant plasmid was transformed into BL21(DE3) for expression .The fusion protein Tat(3861) was expressed with relative molecular weight(MW) 21300 under induction of IPTG, purified by NiNTA column and testified with Two different antiTat rabbit serum and HIVpositive serum by ELISA. The results showed that: ①To exclude the interference of the carrier protein( PET32a), we identified Tat (3861) expressed by the pET32a vector using rabbit antiPEPTIDETat (1101) serum (the two vector sequence are no homology). The result shows that there is a specific reaction and the reactivity much lower than the native Tat, which is in line with expectations. ②To pletely exclude the interference of the carrier protein ( PET32a), we identified Tat (3861) using HIVpositive serum (containing only antiTat antibody). The result shows that both Tat (3861) and the native Tat(1101) have the same specific reaction trend. ELISA detection of the above results suggest that the neutralization epitope of Tat basic region has been preserved by Tat (3861).To conclude, we successfully constructed and expressed Tat(3861) fusion proteins, and its’ neutralization epitope of Tat basic region has been preserved, which laid the foundation on getting the antibodies against the basic region and screening phage display library . 2. Construction of HIV1 tat3861 (51N/55N) basic region mutant libraryStudies have shown that after two important sites（51K and 55R）of the Tat basic region are mutated, the neutralization epitop is not destroyed, and the activity of Tat trans第二軍醫(yī)大學(xué)碩士學(xué)位論文 英文摘要7membrane and extracellular toxicity disappeared or were greatly reduced. In addition, construction of conformable large molecule mutation library is pivotal for molecular evolution study in vitro. In order to extend the range of the screening region of amino acid in Tat basic region gene, the 51K and 55R were mutated randomly by overlap PCR amplification with the random nucleotide primers. At the same time we add to six random linker (SNN) 6 after the basic region 3861, which not only expand the library of variants, but also help to generate a stable conformation of the novel immunogen.Through four rounds of PCR we got the tat3861 (51N/55N) mutant fragments, and cloned into phage display vector pCANTAB5SLD3 after digested by Xba I . then we constructed HIV1 strain Tat3861 (51N/55N) basic region mutant library .The phage displayed library was generated by M13K07 rescue. After detection we found that as much as 106 clones were obtainded in the phage library and the titer was 1012 TU/ml. About 70% clones contained inserted Tat basic region 3861(51N/55N) fragments. Sequence analysis of 14 samples showed that nucleotide acids and amino acids at randomized 51N55N(SNN)6 sites distributed randomly In summary, we have successfully constructed HIV1 strain Tat3861 (51N/55N) basic region mutant library, and its storage capacity, diversity and randomization is all to meet the requirements of library construction.3. Affinity screening of HIV1 tat3861 (51N/55N) basic region mutant libraryThrough the first part of this study we know that the neutralization epitope of Tat basic region has been preserved. By using two kinds of rabbit antisera :PET32Tat (38101) and rabbit antiPET32Tat (1 101), the phage libraries were screened and positive mutant clones were enriched. After two rounds screening we might get the he original sequence of those enriched mutant clones which were stronger affinity, lower transmembrane activity . Each round after the end of screening ,we identified the enriched mutant clones using PCR and sequence analysis.The results of PCR identification showed that: after two rounds screening by using rabbit antiPET32 Tat (1101) serum , from prescreening to screening ,the proportion of the monoclonal containing two basic region fragments in the library is decreased from 56% to 4%, while the proportion of the monoclonal containing single basic region fragment in the library is increased from 39% to 91%。 after two rounds screening by using rabbit antiPET32 Tat (38101) serum , from prescreening to screening ,the proportion of the 第二軍醫(yī)大學(xué)碩士學(xué)位論文 英文摘要monoclonal containing two basic region fragments in the library is decreased from 56% to 0, while the proportion of the monoclonal containing single basic region fragment in the library is increased from 39% to 87%. In theory, the monoclonal containing two basic region fragments could easily be obtained through evolutionary selection because of a selective advantage. It seems to suggest that the mutant sites 51N55N(SNN) 6 is playing a more important role for containing the neutralization epitope of Tat basic region of large than repeated fragments. The results of sequence analysis showed that: ①after two rounds screening by using rabbit antiPET32 Tat (1101) serum , ratio of the reverse connecting between basic fragment and the vector is up to 50% .while the ratio is only 6% after two rounds screening by using rabbit antiPET32 Tat (38101) serum. It seems to suggest that antiPET32 Tat (38101)