Construction of Larval cDNA Expression Library and Immunological Identification of Positive Clones in Tick Haemaphysalis qinghaiensis

A primary cDNA expression library with a titer of 5.0 × 105 PFU mL-1 was constructed from mRNA extracted from larval Haemaphysalis qinghaiensis ticks in order to identify certain genes,which would then be used as candidate molecules for development of effective vaccines to control this parasite.Totally 11 positive clones,which designated as HqL01-11,were obtained by immunoscreening of the library using a polyclonal antibody generated in rabbit with larval tick protein extract.Results of sequence analysis from BLASTN searching revealed that 6 of them had no significant homology with the adult H.qinghaiensis ticks’ known genes,4 of them had no significant homology with all genes deposited in GenBank database.HqL07,HqL08,HqL09,and HqL11 were deposited to GenBank database,and accession numbers were EF605263,EF605264,EF605265,and EF605266,respectively.Subsequently,HqL07 and HqL09 were expressed in vitro and the molecular weights of the corresponding expressed products were 60 and 70 kDa,respectively.Western blot analyses showed that HqL07 and HqL09 had immunogenicity.This study laid the foundation for future production of genetically engineered vaccines for the immunological control of H.qinghaiensis.

Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon

mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyAT-tract System 1000 Kit. By using mRNA as template, double - strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR l/Xho Ⅰ, and the recombinant DNA was in vitro packaged into lambda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XL1 - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 10~5pfu in capacity and its recombination ratio is higher than 99 % . The size of the inserted cDNAs was determined by EcoR l/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus monodon hepatopancreas and is available for screening and expression of shrimp genes.

Cloning and screening of cDNA of Psilgramma menephorn allergen

Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

Cloning of Novel Tumor Metastasis-Related Genes from the Highly Metastatic Human Lung Adenocarcinoma Cell Line Anip973

A cDNA library was successfully constructed from Anip973, a human lung adenocarcinoma cell line with high metastatic potential. NIH3T3 cells were stably transfected using this cDNA library and screened for morphological changes in a soft agar assay. Genomic DNA was isolated from putative clones and the integrated sequence was retrieved by PCR and sequencing. Three known genes, ribosomal protein L23, hypothetical protein FLJ22104, and serine protease inhibitor, kazal type 6 and a number of 5＇-terminally truncated sequences were identified. Furthermore, cells transfected with ribosomal protein L23 was highly invasive compared with the empty vector as control （P 〈 0.02）. These results indicate that the expression cloning of cDNA libraries in NIH3T3 cells and subsequent screening for loss of contact inhibition in soft agar is a viable tool for identifying tumor-related genes and ribosomal protein L23 gene plays a role in cell movement and metastasis.

Generation and analysis of 113 adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags

To rapidly and economically obtain knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes using expressed sequence tag (EST) Methods A directional cDNA library constructed from Schistosoma japonicum (Chinese strain ) adult stage RNA was used to generate expressed sequence tags(ESTs) These were compared against an EMBL parasites database and GENBANK database by BLASTn and BLASTx Results A total of 314 phage clones were randomly selected for generating expressed sequence tags(ESTs) From these clones, 132 EST quality sequence were obtained Among these EST quality sequences,113 ESTs were successfully submitted to the dbEST at GenBanK A total of 7 6% of these EST quality sequences were previously identified sequence of Schistosoma japonicum, while 4 5% were putatively identified sequences of Schistosoma japonicum A total of 23 5% of these EST quality sequences were putatively identified sequence of Schistosoma mansoni or other organisms 57 6% had no matches in the database and were classified as unknown sequences Most ESTs with the putative protein identified belonged to housekeeping proteins Information about several interesting genes was found Conclusion Partial cDNA sequencing to generate expressed sequence tags (ESTs) has the potential to rapidly and economically increase our knowledge about adult stage Schistosoma japonicum(Chinese strain) expressed genes