Objective To construct human myeloma cell cDNA expression library as to screen myeloma tumor antigen. Methods Total RNA and purified mRNA were extracted from human myeloma cell line HMy2. First and second strand cDNA ...Objective To construct human myeloma cell cDNA expression library as to screen myeloma tumor antigen. Methods Total RNA and purified mRNA were extracted from human myeloma cell line HMy2. First and second strand cDNA were synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested by Xho I, and smaller than 400bp were removed by Sephacryl-S400 spin column, the remaining were ligated with λZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E.coli XL1-Blue-MRF for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid were excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain , and then the pBK-CMV phagemid were digested by Xho I and EcoR I. Results The HMy2 cell line cDNA library consisting of 1.58×10 6 recombinant bacteriophages was constructed with the recombinant ratio 99.6%. The average length of the recombinant exogenous inserts was about 1.7kb.Conclusion The constructed cDNA library are deserved to screen target clones.展开更多
Objective To construct a λgt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Methods Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA pu...Objective To construct a λgt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Methods Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 μg) was submitted to reverse transcription using random hexamers ［pd(N6)］. The double-strand blunt-ended cDNAs were ligated with an EcoRI/NotI adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector λgt11 EcoRI arms. After being packaged in vitro, λgt11 was put to an infectious bacteria Echinococcus coli (E.coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments.Results The recombinant ratio was nearly 100% and approximately 1×106 clones could be derived from this λgt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. Conclusions A λgt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.展开更多
文摘Objective To construct human myeloma cell cDNA expression library as to screen myeloma tumor antigen. Methods Total RNA and purified mRNA were extracted from human myeloma cell line HMy2. First and second strand cDNA were synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested by Xho I, and smaller than 400bp were removed by Sephacryl-S400 spin column, the remaining were ligated with λZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E.coli XL1-Blue-MRF for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid were excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain , and then the pBK-CMV phagemid were digested by Xho I and EcoR I. Results The HMy2 cell line cDNA library consisting of 1.58×10 6 recombinant bacteriophages was constructed with the recombinant ratio 99.6%. The average length of the recombinant exogenous inserts was about 1.7kb.Conclusion The constructed cDNA library are deserved to screen target clones.
文摘Objective To construct a λgt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Methods Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 μg) was submitted to reverse transcription using random hexamers ［pd(N6)］. The double-strand blunt-ended cDNAs were ligated with an EcoRI/NotI adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector λgt11 EcoRI arms. After being packaged in vitro, λgt11 was put to an infectious bacteria Echinococcus coli (E.coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments.Results The recombinant ratio was nearly 100% and approximately 1×106 clones could be derived from this λgt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. Conclusions A λgt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.
