Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B

摘要

Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRJZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS HI/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to XTriplEx2 vector. Then sigma phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional liter determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The liters of unamplifed and amplified libraries were 1.94x10~6 pfu/ml and 1.49xl09 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15 percent in unamplified library and 98.76 percent in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29 percent , and 0.5-1.0 kb in 35.71 percent. Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.