Cloning, Expression Of L1 Reverse Transcriptase Gene In Human Multiple Myeloma Cell Line ARH-77 And Production Of The Polyclonal Antibodies

Over 50 000 LI sequences exist in human genome, which accounts for as much as 15 % of human genome, 4 000 of these are full length, whereas the rest are truncated at the 5' end. LI lacks long terminal repeats and is characterized by a poly(A) tail.A full-length mammalian LI sequence contains a 5' untranslated region with a internal promotor, two nonoverlapping open reading frames (ORF I and ORFII ) ,and a 3' untranslated region that ends in a poly(A) tail. ORF I encodes a RNA-binding protein, whereas ORF II encodes an endonuclease-reverse transcriptase activity and a cysteine rich domain.In this study, a 583 bp segment of L1 reverse transcriptase gene was amplified by PCR using human multiple myeloma cell line ARH-77 cDNA library as a template. 5' end of the DNA sequence of L1 reverse transcriptase gene was obtained by in situ hybridization using L1 reverse transcriptase gene as a probe. Total RNA from the ARH-77 cell was isolated and obtained 3' end and poly (A) of L1 reverse transcriptase gene by RT-PCR.We compared the sequences of #6 positive clones screened by in situ hybridization and the sequences obtained by RT-PCR, and analysed and spliced to obtain the DNA sequence of L1 reverse transcriptase gene. According to analysing by ORF finder and Conserved Domain Search in NCBI, the L1 reverse transcriptase gene in human multiple myeloma cell line ARH-77 has an open reading frame with 552 base pairs and encodes a 184 amino acid polypeptide with a theoretical 21 kD.A 552 bp sequence of L1 reverse transcriptase domain was acquired by PCR, then recombinant prokaryotic expression plasmid pQE30-L1 ORF was constructed by inserting LI ORF into prokaryotic expression vector pQE30. The expression of the recombinant plasmids was induced with 1 mmol/L IPTG. SDS-PAGE electrophores analysis showed that a new protein with molecular weight 21kD could be found. The expression level of L1 fusion protein was increased when IPTGconcentration was increased and would reach its highest expression at 6h.The prokaryotic expression vector pQE30 is fusion protein expression vector in which the target protein was expressed have been tagged with 6 consecutive histidine residues (6 xHis tag) in the N-terminal. The use of the affinity tags simplifies the purification of L1 fusion proteins by employing affinity chromatography methods.A rabbit was used to produce polyclonal antibodies of L1 fusion protein. In the first immune experiment, L1 fusion protein was blended with a same volume of Freund's complete adjuvant and injected into the back of the rabbit by subcutaneous injection. In the rest of the immune experiments, L1 fusion protein was amalgamated with Freund's incomplete adjuvant and injected into the back of the rabbit. After 4-7 times, polyclonal antibodies were produced and testified by immune diffusion and ELISA.