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

• Published in: Journal of Zhejiang University. B. Science Vol. 6; no. 4; pp. 288 - 294
• Main Author: 陈晓红 陈智 姚航平 陈峰 朱海红 周红娟
• Format: Journal Article
• Language: English
• Published: China Institute of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China%Department of Basic Medicine, School of Medicine, Zhejiang University, Hangzhou 310003, China 01.04.2005; Zhejiang University Press
• Subjects: Biomedicine; cDNA; DNA, Complementary - genetics; Gene Library; Hepatitis B, Chronic - genetics; Humans; Liver - metabolism; Liver - pathology; Polymerase Chain Reaction; RNA - analysis; RNA - genetics; 慢性乙型肝炎; 治疗方法; 肝脏组织; Chronic hepatitis B; Human liver tissue; Construction and characterization; cDNA library
• ISSN: 1673-1581; 1862-1783
• DOI: 10.1631/jzus.2005.b0288

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 TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS Ⅲ/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 λTriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×10^6 pfu/ml and 1.49×10^9 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.