Appling RD-PCR Technique To Construct HIV Random Peptide Library

The large amounts of DNA sequence data in genomics projects has led to an increasing demand for powerful tools to analyze protein function and interaction. Library construction is one of the most important method in dissociate genes of interest so as to investigate clone by clone of the encoding sequences as well as protein expression. cDNA libraries and genomic fragment libraries have show promising results in genome wide protein interaction screening. To further explore the proteomics, small fragments library should be constructed, as it may be more compatible with secretion machinery and less prone to aggregation due to mis-folding. Moreover, fragments libraries can help to identify protein interaction based on domain and motif. Yeast two-hybridization approaches having been demonstrated that fragments libraries play important role in protein interaction research. A different but novel library technique was studied, which is based on RD-PCR technology.Restriction display PCR (RD-PCR) technique offers an effective method for molecular research. It provides reproducible and highly representative molecular snapshots of genes expressed in various cells. Besides, it has been widely applied in sample-label and preparation of DNA fragments for cDNA microarray probes. RD-PCR technique used as followed: The genome DNA or cDNA was cleavaged into fragments by restriction endonuclease Sau3A I. Adapters designed by research needs were linked to both ends of the fragments, then the universal primers complementing to specific sequence of the adapters were used as primers to amplify DNA fragments. Here we applied RD-PCR to constructing fragments expression library by using threeframeshift adaptors. The three adapters shared almost the same sequence except for one base increment one by one. Three adapters randomly ligated to both side of each digested fragment, rendering 9 groups of different ligation products. Then PCR were performed to amplify ligation products. After this, DNA fragments were cloned to expression vector to construct genome fragments library. Because every fragment can insert vector directly or in reverse, each fragments with 9 different liagation styles could totally express 18 frame shift peptides. Among these peptides, there is one that should be the predicted target peptide.HIV virus genome was used as testing model to validate RD-PCR technology in constructing peptides library, as its overlap gene was compatible with frame shift adapter design. And the full length of HIV subtype B in U26942 is only 9000 base pairs; therefore, the constructed library could be analyzed more systematically.In order to testify the flexibility of adapter design, we designed two groups of adapters (each have three frame shift adapters) and relevant primers according to two vectors: one is prokaryotic expression vector pET22b, the other is yeast expression vector pMNT-TOPO. Moreover, experimental conditions of RD-PCR were optimized. Under the best conditions (adapters and fragments ligation ratio was between 30:1 to 45:1, while RD-PCR annealing temperature was ladderlike), two HIV genome libraries with different group of adapters were constructed.First of all, fragment libraries were screened for quality analysis. 48 and 32 random clones were isolated respectively from the two DNA librarie. The results of sequence showed that, most of 48 clones chosen from prokaryotic adapter library had full primers and adapters sequence, which coincided with what had been expected. The ligate probability of the three adapters Apl > Ap2^ Ap3 was 34.9%, 31.4%, 33.7 %, respectively. The probability of directly insertion is 55.8%. Except for the loss of 104 bp and 441bp fragments caused by random factor, the other fragments longer than lOObp were all involved in. The result of yeast vector library was comparable with that of the prokaryotic vector library. It was found that clones contains the digested fragments(all of the fragments longer than lOObp, mostly of the fragments with 3OObp length), the ligation probability of the three adapters were Ayl -, Ay2% Ay 3 was 35%, 31.7%, 33.3%, respectively. The sequence of the adapter and primer consisted with expected result, and the direction of inserting was verified. The resultshave demonstrated that adapters and primers have been designed successfully. The fragment libraries of HIV genome constructed by RD-PCR could be used for further studies.After obtaining HIV genome DNA library in prokaryotic vector and yeast vector, we commenced construction of expression library. We built the HIV fragments expression library with prokaryotic vector library for the following reasons. First, there were less repeat segments in the DNA library with adapters designed by pET22b vector, for that we could get more gene informations in a given library. Second, expression proteins in prokaryote were comparatively less complicated to manipulate.The products of RD-PCR were digested by Not I and ligated to pET22b vector, then transformed to competent cell (XL-1). In order to analyze the expression of polypeptide, we singled out 80 clones, which were identified by PCR and analyzed by sequencing. The PCR result showed 66 positive clones, the ratio is 82.5%. Among the 66 clones, 5 of them were pET22b vectors, and others were recombined plasmids, which were inserted by HIV DNA fragments. 32 clones contained reverse inserted fragments, and these recombined plasmids couldn't express HIV protein, they could only express random polypeptide. The rest clones containing directly insected DNA segments were analyzed by DNASTAR Editseq. Clone H10, B04 and C04 which could express HIV peptide were gained, subsequently being expressed in E.coli BL21(DE3). The SDS-PAGE results showed that only plasmid H10 could highly express protein, which could also be testified positive by western blot with anit-His6 antibody. From all of these, we could find that the expression library constructed included not only the peptide of HIV protein, but also random expression fragments formed by the reversed insert and ORF frameshift due to adapters.In summary, RD-PCR technique offers a new method for constructing fragments library, which is essential for study the interaction between proteins and peptides. And the genome fragment expression library constructed by RD-PCR technique is a random peptide library, among which the genome self-peptide was expressed.