DEVELOPMENT AND COMPARISON OF POTENTIAL HIV-TRANSMISSION PREVENTION PROTEINS NATIVE TO FRESHWATER CYANOBACTERIA

Open Access
Author:
Jones, Ryan Joseph
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wayne Roger Curtis, Thesis Supervisor
  • Scott Thomas Milner, Honors Advisor
Keywords:
  • HIV
  • cyanobacteria
  • cyanovirin
  • microvirin
  • cyanobacteria transformation
  • Microcystis aeruginosa
  • anti-retroviral
  • microbicide
  • molecular cloning
  • Intein
  • Solubility tag
  • Fusion cloning
  • Cyanobacteria PCR
Abstract:
As cases of Human Immunodeficiency Virus (HIV) continue to rise amongst adolescents and young adults in sub-Saharan Africa, the need for additional support becomes more urgent. Particularly in developing nations, access to anti-retrovirals is highly dependent on socioeconomic and psychological barriers that are not easily breached. Alternatively, there exists a lectin protein, cyanovirin-n (CVN), that prevents the transmission of HIV through inhibition of the gp120 and CD4+ receptor complex. This work sought to develop a platform to clone, express, and purify cyanovirin for end use in a topical anti-viral. CVN was successfully cloned into a cyanobacteria transformation vector, but could not transform Synechococcus 7002 due to apparent toxicity. In an effort to alleviate this toxicity, a cloning strategy was developed to fuse the CVN gene to an aggregation tag to alter its folding properties and reduce cellular toxicity upon expression. In tandem, a strategy was developed to isolate Microcystis aeruginosa from algal blooms in freshwater bodies near Lake Wallenpaupack in Northeast Pennsylvania. This cyanobacteria produces microvirin (MVN), a similar protein to CVN exhibiting less in vivo toxicity. Attempts to amplify the MVN gene from these water samples were unsuccessful; however, to facilitate future success of this project, a procedure to clone genes from cyanobacteria culture has been developed. Additional progress towards cloning MVN has involved isolation of Microcystis aeruginosa by serial dilution plating and non-photosynthetic contamination testing. Successful fusion of these anti-HIV transmission proteins to aggregation tags would provide a cost-effective and scalable platform for production of antiviral microbicides in Synechococcus.